Hybrid drug combination: Combination of ferulic acid and metformin as anti-diabetic therapy

ABSTRACT
Background and Purpose: Ferulic acid, an anti-oxidant phytochemical present in several dietary components, is known to produce wide range of pharmacological effects. It is approved for use in food industry as a preservative and in sports food. Previous reports from our lab have shown synergistic interaction of ferulic acid with metformin in cell lines and diabetic rats. The purpose of this review is to compile information about anti-diabetic activity of ferulic acid in in vitro and in vivo models with special emphasis on activity of ferulic acid when combined with metformin. The mechanism of synergistic interaction between ferulic acid and metformin is also proposed after carefully studying effects of these compounds on molecules involved in glucose metabolism.Methods: Scientific literature for the purpose of this review was collected using online search engines and databases such as ScienceDirect, Scopus, PubMed and Google scholar.Results: Ferulic acid forms resonance stabilized phenoxyl radical which scavenges free radicals and reduce oxidative stress. It improves glucose and lipid profile in diabetic rats by enhancing activities of antioxidant enzymes, superoxide dismutase and catalase in the pancreatic tissue. Combining ferulic acid with metformin improves both, in vitro glucose uptake activity and in vivo hypoglycemic activity of the latter. It is possible to reduce the dose of metformin by four folds (from 50 to 12.5 mg/kg body weight) by combining it with 10 mg of ferulic acid /kg body weight in diabetic rats. Ferulic acid improves glucose uptake through PI3-K pathway whereas metformin activates AMPK pathway to improve glucose uptake.Conclusion: The synergistic interaction of ferulic acid and metformin is due their action on parallel pathways which are involved in glucose uptake. Due to synergistic nature of their interaction, it possible to reduce the dose of metformin (by combining with ferulic acid) required to achieve normoglycemia. Since the dose of metformin is reduced, the dose associated side effects of metformin therapy can be reduced.

1.Introduction
Diabetes mellitus (DM) is a group of heterogeneous metabolic disorders characterized by increased blood sugar (hyperglycemia) due to reduced insulin secretion and/or reduced activity of it at various target sites such as liver, skeletal muscles and adipose tissue (Nolan et al., 2011). If the hyperglycemia associated with disease is not controlled, it leads to overproduction of reactive oxygen species (ROS), which in turn are responsible for β-cell dysfunction (Drews et al., 2010) and other secondary complications (Giacco et al., 2010). ROS are also implicated in the development of insulin resistance (Houstis et al., 2006) and so they are both, cause and consequence of diabetes.Polyphenolic compounds are widely present in food and beverages and in addition to their several beneficial properties; they are also known to be strong antioxidants. The anti-diabetic activity of polyphenolic antioxidant phytochemicals in various in vitro and in vivo models is compiled in a review article elsewhere (Vinayagam et al., 2016).

2.Ferulic acid: A potent antioxidant phytochemical
Ferulic acid (4-hydroxy,3-methoxy cinnamic acid) (Fig. 1), an organic monophenolic phytochemical, is commonly found in our every day food including grains (40-90 mg/100 g) such as wheat, rice and oats, vegetables (6-30 mg/100 g) such as tomato, eggplant, broccoli and spinach, fruits (1-10 mg/100 g) such as strawberries, banana and citrus, and beverages such as coffee (1-10 mg/100 g) and beer (0.2-0.9 mg/100 g) (Mancuso et al., 2014; Westfall et al., 2015; Zhao et al., 2008). In addition, ferulic acid is also present in Chinese medicinal herbs including Cimicifuga racemosa, Angelica sinensis, and Ligusticum chuangxiong (Ou et al., 2004). Ferulic acid occurs as free form or in conjugation with hydroxyl acids (in vegetables and fruits) or hemicellulosic polysaccharides (in grains) (Mathew et al., 2004; Zhao et al., 2008).Ferulic acid forms phenoxyl radical which is resonance stabilized due to the presence of methoxy and carboxylic acid groups in it. The phenoxyl radical is mainly responsible for the free radical scavenging activity of ferulic acid (Paiva et al., 2013). In addition to scavenging ROS, ferulic acid is also reported to increase the expression levels of anti-oxidant enzymes, superoxide dismutase (SOD) and catalase. Ferulic acid is observed to restore the reduced levels of SOD and catalase in pancreatic tissue in streptozotocin (STZ) induced rats (Roy et al., 2013). Sodium salt of ferulic acid is reported to improve the levels of SOD in myocardium of STZ-induced rats (Xu et al., 2012). Ferulic acid has also been reported to modulate insulin signaling molecules in high- fat diet and fructose-induced type-2 diabetic adult male rats. The diabetic animals treated with ferulic acid shows downregulation of gluconeogenic enzyme genes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), thereby reducing gluconeogenesis (Narasimhan et al., 2015).

Ferulic acid is reported to show very low toxicity even after administration of very high doses in rodents. The LD50 value in rats was observed to be higher than 2 g/kg body weight in rats (Ou et al., 2004). Ferulic acid has been approved for usage in food industry. Food and cosmetic grade ferulic acid is available commercially. Due to its antioxidant and antimicrobial properties, it is approved as food preservative in Japan. It is also used in sport food to enhance athletic performance (Ou et al., 2004). A sodium salt of ferulic acid, sodium ferulate, is used in traditional Chinese medicine for the treatment of cardiovascular diseases (Wang et al., 2005).It is reported that the antioxidant activity of phenolics is dependent on the number and location of their phenolic groups and hence different phenolic compounds show different biological activities (Amic et al., 2007). The change in the number and position of phenolic groups would also affect their binding to target protein which would result in change in their biological activity.

3.Anti-diabetic activity of ferulic acid
Ferulic acid has been shown to have anti-diabetic effect in various in vitro and in vivo model systems. Treatment of STZ induced diabetic rats with ferulic acid (50 mg/kg body weight, orally) for 8 weeks improved the blood glucose levels and other biochemical parameters such as serum triglycerides, total cholesterol, urea and creatinine (Roy et al., 2013). The main feature of STZ induced diabetic rats is increased oxidative stress in pancreatic islets which causes their necrosis leading to reduced secretion of insulin (Lenzen, 2008). Treatment with ferulic acid resulted in improvement in the activities of antioxidant enzymes, SOD and catalase, and reduced glutathione in the pancreatic tissue (Roy et al., 2013). Oral administration of 10 mg/kg body weight of ferulic acid in alloxan induced diabetic mice for 15 days improved the disturbed levels of serum glucose, urea, uric acid and creatinine. The levels of antioxidant markers were found to be enhanced in kidney, liver and serum in ferulic acid treated mice (Ramar et al., 2012).
Ferulic acid has also seen to improve blood glucose and lipids in high fat diet fed obese rats (Song et al., 2014). Purified ferulic acid from the leaves of Hibiscus mutabilis has shown to improve palmitate induced insulin resistance in L6 myotubes. The purified compound also reduced the blood glucose to normal in high fat diet fed rats following 15 days of oral administration (Gogoi et al., 2014). Ferulic acid purified from stems of Cucurbita moschata improved insulin stimulated glucose uptake in insulin resistant FL83B cells (normal mouse hepatic cell line). The cells were made insulin resistant by incubating them with 20 ng/mL of TNF-α (Chang et al., 2014). So these studies clearly indicate that ferulic acid has the potential to improve the insulin resistance. So combining ferulic acid with insulin can result in improved glycemic control.

4.Synergy between ferulic acid and metformin
Metformin (1,1-dimethylbiguanide) (Fig. 1), a biguanide, is a first line agent used in the treatment of type 2 diabetes. The mechanism of action of metformin is not fully understood but the role of AMP-activated protein kinase (AMPK) has been implicated in its glucose lowering activity (Zhou et al., 2001). Liver is the major site of action for metformin. It controls the hyperglycemia by suppressing the hepatic glucose output by the inhibition of gluconeogenesis. It also reduces the blood sugar by improving glucose disposal and glycogen synthesis in skeletal muscles (Setter et al., 2003; Stumvoll et al., 1995). The metformin therapy is associated with side effects such as lactic acidosis, diarrhea, abdominal pain, nausea, anorexia, and flatulence (Setter et al., 2003). Most of the side effects of it are dose associated and reducing its dose by combining with other drugs or with a phytochemical can reduce the side effects.Combined effect of ferulic acid and metformin on 2-deoxyglucose (2-DG) uptake in 3T3L1 adipocytes and L6 myotubes is systematically studied using an isobologram method (Prabhakar et al., 2009). Here 2-DG, a non-metabolisable analog of glucose, is used to measure the glucose uptake rate. Treatment of L6 myotubes with 15 μM of metformin resulted in 219 ng of 2-DG uptake by 3.5 x 105 cells. While the same amount of 2-DG uptake was achieved with a combination of 5 μM of ferulic acid and 5 μM of metformin or 10 μM of ferulic acid and 3 μM of metformin indicating synergy (Fig. 2). So the dose of metformin can be reduced by 3 to 5 folds by combining it with about 5 to 10 uM of ferulic acid (Prabhakar et al., 2009). Similar synergistic addition of 10 μM of ferulic acid to 5μM of metformin reduced the dose of metformin by four folds to achieve 237 ng uptake of 2-DG by 3.5 x 105 3T3L1 cells (Prabhakar et al., 2011).

The combined effect of metformin and ferulic acid is studied in STZ induced diabetic rats (Prabhakar et al., 2013). When used individually for 21 days, metformin at a dose of 50 mg/kg body weight and ferulic acid at a dose of 40 mg/kg body weight reduced the blood sugar by two folds when compared to untreated diabetic rats (Prabhakar et al., 2013). Another study reported treatment of STZ induced diabetic rats with ferulic acid alone at a dose of 50 mg/kg body weight for 56 days reduced the blood glucose by three folds when compared to untreated diabetic rats (Roy et al., 2013). The combinations of ferulic acid (10 mg/kg body weight) with metformin (12.5 mg/kg body weight) significantly reduced blood sugar level over their individual treatments (Prabhakar et al., 2013). It was possible to achieve a four fold reduction in the dose of metformin (from 50 to 12.5 mg/kg body weight) in the combination study by adding 10 mg of ferulic acid /kg body weight.Ferulic acid and metformin improved lipid profile in diabetic rats but the combination did not offer any added advantage over individual treatment. Treatment of diabetic rats with ferulic acid, metformin or their combination reduced the level of cholesterol, very low density lipoproteins and triglycerides by 1.5 folds. All the treatments decreased level of low density cholesterol by1.75 folds in diabetic rats. Combined treatment of ferulic acid and metformin improved pancreatic β-cell mass significantly over individual treatments. Islet number in pancreas decreased to 4 in diabetic animal. Treatment with metformin (50 mg/kg body weight) alone increased the islet number to 7 whereas that with ferulic acid (40 mg/kg body weight) alone increased the number to 8. Combined treatment of metformin (12.5 mg/kg body weight) andferulic acid (10 mg/kg body weight) increased the number to 10 (Prabhakar et al., 2013). The combination treatment improved β-cell regeneration in diabetic rats even at lower doses of metformin. Ferulic acid, being an anti-oxidant, reduces the oxidative stress induced by STZ in pancreatic β-cells (Roy et al., 2013) while, metformin, being an insulin sensitizer, reduces the amount of insulin required to reduce the blood sugar and so decreases the burden on pancreatic β-cells. So when given together, metformin and ferulic acid improve hyperglycemia and β-cell dysfunction in STZ induced diabetic rats (Prabhakar et al., 2013).

5.Mechanism of synergy between ferulic acid and metformin
Glucose uptake in myotubes and adipocytes is mainly mediated by GLUT4 transporter. It is proposed that (Prabhakar et al., 2011; Prabhakar et al., 2009) ferulic acid improves 2 DG uptake in L6 myotubes and 3T3L1 adipocytes through PI3-K (phosphatidylinositol-3 kinase) dependant pathway by increasing the expression levels of PI3-K and GLUT4 (Fig. 3). Metformin is also reported to increase expression levels of GLUT4 (Prabhakar et al., 2011; Prabhakar et al., 2009). It is reported that treatment of type 2 diabetic patients with metformin for 10 weeks caused increased activity of AMPK in skeletal muscles (Musi et al., 2002). Activation of AMPK has been reported to increase glucose uptake in muscles (Bergeron et al., 1999). Metformin, being an activator of AMPK, improves 2-DG uptake by AMPK dependant pathway. So, ferulic acid acts through PI3-K pathway while metformin acts through AMPK dependant pathway to improve glucose uptake (Fig. 3). Two compounds can cause synergistic interaction when they affect proteins/enzymes on parallel pathways (Agarwal et al., 2012). So it can be proposed that the synergistic interaction between ferulic acid and metformin is due their action on two different pathways which ultimately cause increased uptake of glucose.

6.Pharmacokinetics of ferulic acid
Ferulic acid is reported to be absorbed completely in rat stomach into the blood stream. Ferulic acid has a pKa of 4 and it is suggested that this low value keeps it in the undissociated form which facilitates its transport across the gastric membrane via passive diffusion (Zhao et al., 2004). It disappears quickly from isolated rat intestine indicating its rapid transport across the intestinal membrane (Spencer et al., 1999). However the extent of passive diffusion in this case would be low as ferulic acid does not maintain undissociated form in neutral or weakly acidic conditions. Several transport systems such as Na+-dependent saturable transport mechanism (Wolffram et al., 1995), monocarboxylic acid transporter (Konishi et al., 2002), H+-dependent transport system (Itagaki et al., 2005) have been implicated for its movement across the intestinal membrane. After oral administration in rats, less than 1% of ingested dose was found in feces indicating its complete absorption (Zhao et al., 2003a). Due to the presence of hydroxyl groups, ferulic acid extensively undergoes phase 2 metabolism (conjugation reactions) in liver. Glucoronic acid and sulphate conjugates of ferulic acid are found to be the major metabolites in plasma and urine of rats (Rondini et al., 2002; Zhao et al., 2003b). Ferulic acid and its conjugates are mainly excreted from the kidney in rats (Adam et al., 2002).A few clinical pharmacokinetics studies reported for ferulic acid in humans indicate that these parameters are different in human and rats. Ferulic acid is found to be rapidly absorbed after oral administration (Yang et al., 2007). When given in the form of wheat bran, ferulic acid appears in the free form and its glucuronic acid conjugate in plasma and free form and its glycine conjugate in urine (Kern et al., 2003). Due to extensive metabolism, ferulic acid is reported to have very low oral bioavailability in humans (20%) and rats(9-20%) (Mancuso et al., 2014). Ferulic acid and metformin (at a weight ratio of 1:1) shows synergistic interaction in Wistar rats in controlling blood glucose, but the phytochemical has different pharmacokinetic properties in humans and hence the ratio of these two compounds to achieve the same biological activity may vary in humans.

7.Conclusion
Combining ferulic acid with metformin improved both, in vitro glucose uptake activity and in vivo hypoglycemic activity of the latter. It was possible to reduce the dose of metformin by four folds (from 50 to 12.5 mg/kg body weight) by combining it with 10 mg of ferulic acid /kg body weight. Since the dose of metformin is reduced, the dose associated side effects of metformin therapy (lactic acidosis, diarrhea, abdominal pain, nausea, anorexia, and flatulence) can be reduced by combining it with ferulic compound 3k acid.