Uses, Adverse Drug Reactions and Drug Interactions of Metformin

## **Chapter 4** Metformin: Pros and Cons

*Shalini Sivadasan, Muthukumar Subramanian and Rajasekaran Aiyalu*

#### **Abstract**

Metformin was approved for the treatment of Type 2 Diabetes Mellitus in 1958 for UK, in 1972 for Canada and in 1995 by FDA in USA. Metformin is the drug of choice for patients who are obese and have type 2 diabetes mellitus. Though metformin was at first proven to treat hyperglycemia, many other uses of metformin are proven to be effective. It is also used for gestational diabetes mellitus, obesity, hyper secretion of ovarian androgen, poly-cystic ovary syndrome (PCOS), anti-psychotic therapy induced weight gain, cancer treatment and anti-aging. Metformin causes a decrease in appetite thus known to act on obesity. The other action of metformin is reduction of circulating levels of insulin and insulin like growth factor 1 (IGF-1) which is associated with anticancer action. There are ongoing researches about the effect of metformin on anti-aging properties and proved that metformin is linked with anti-aging factors. Three main factors that are related with aging are oxidation, glaciation and methylation. Metformin as all drugs, have unwanted effects as well. Many side effects of metformin are considered mild where lactic acidosis and vitamin B12 deficiency happens to be the major.

**Keywords:** metformin uses, diabetes mellitus, obesity, poly-cystic ovary syndrome, cancer

#### **1. Introduction**

#### **1.1 Pros (uses) of metformin**

Metformin, the most common drug used to treat type 2 diabetes, approved by U.S. Food Drug Administration (US-FDA) (1), belongs to a class of drugs called biguanides with a guanidine and galegine connection. Metformin was approved for treatment of Type 2 Diabetes Mellitus in 1958 for UK [1], 1972 for Canada [2] and 1995 by FDA in USA [1, 3].

Metformin (1,1-dimethyl biguanide hydrochloride) was synthesized in 1920's. Since then, the drug became the first choice to treat type 2 diabetes due to its remarkable ability to decrease plasma glucose levels [4–6]. It acts by reducing the glucose made by liver, decreasing the amount glucose that body absorbs and increasing the effect of insulin in the body [7].

In recent years, studies have shown many unexpected effective roles of metformin that exerts strong effect on cardiovascular disease (CVD) [8], cancers [9, 10], neurodegenerative diseases [11], liver diseases [12], obesity [13, 14], and renal diseases [15], hypersecretion of ovarian androgens, poly-cystic ovary syndrome (PCOS), anti-psychotic therapy induced weight gain, and anti-aging [16]. The agent also offers neuro protection that may reduce the risk of dementia and stroke [17].

#### *1.1.1 Metformin in diabetes*

Several studies and clinical trials have confirmed that metformin mono therapy or combination therapy with other glucose-lowering drugs is successful in treating type 2 diabetes. Metformin is the drug of choice for diabetic patients who are obese and have type 2 diabetes mellitus.

Type 2 diabetes coexists with insulin resistance and leads to extremely high blood sugar levels. Metformin lowers blood sugar, preventing permanent organ damage, which in due course could lead to dysfunction and failure [18, 19]. Metformin exerts its anti-hyperglycemic effects through AMP which initiates the uptake of sugar from the blood into muscles.

Metformin exerts its anti-hyperglycemic effects by suppressing hepatic glucose production through AMPK dependent [20, 21] or -independent pathways [22, 23].

Metformin increases AMPK that leads to more sugar being taken from the blood into tissues, thus lowering the blood sugar level [24].

It is used in case of insulin resistance as it works by decreasing hepatic glucose production, decreasing peripheral insulin resistance and improving insulin sensitivity thereby increasing peripheral glucose uptake and utilization. Metformin does not produce hypoglycaemia and does not cause hyperinsulinemia in normal patients or in patients with type 2 diabetes. Insulin secretion remains unchanged whereas fasting insulin levels and daylong plasma insulin response may decrease with metformin therapy [25].

On the other hand, metformin may reduce blood sugar by inhibiting the production of new glucose (gluconeogenesis) from non-carbohydrates such as lactate, glycerol, and some amino acids [23]. Metformin inhibits gluconeogenesis through AMPK-dependent activation of small hetero dimer partner (SHP) and inhibition of phosphorylation of CREB binding protein (CBP) [26], thereby suppressing the expression of gluconeogenic genes, such as G6Pase (glucose 6 phosphatase), PEPCK (phosphoenolpyruvate carboxykinase), and PC (pyruvate carboxylase) [27].

Studies also suggest that metformin could enhance GLUT1 (glucose transporter 1) mediated glucose transport into hepatocytes by activating IRS2 (insulin receptor substrate two), decreasing plasma glucose levels [28].

Besides decreasing liver glucose production, metformin also decreases glucose levels through increasing (i) GLUT4 (glucose transporter 4) mediated glucose uptake in skeletal muscles [29] and (ii) absorption of glucose in the intestines [30]. Metformin also stimulates glucagon-like-peptide-1 (GLP-1) release, thereby improving insulin secretion and reducing plasma glucose levels [31]. The molecular mechanism of metformin in hepatic gluconeogenesis and glucose production is shown in **Figure 1**.

A clinical trial conducted on over 3,000 people who were at risk of developing type 2 diabetes showed that those people treated with metformin had a 31% lower occurrence of type 2 diabetes compared to the placebo group [34].

#### *1.1.2 Metformin in gestational diabetes*

Gestational diabetes mellitus (GDM) is the most common medical complication of pregnancy which is associated with insulin resistance (IR) and hyperinsulinemia that may predispose some women to develop diabetes. Gestational diabetes has been defined as any degree of glucose intolerance with an onset, or first recognition during pregnancy [35]. In 2013, the World Health Organization (WHO) recommended

#### **Figure 1.**

*Metformin in hepatic gluconeogenesis and glucose production. Metformin acts primarily to suppress glucose production in the liver. While metformin's mechanism(s) of action remain controversial, current evidence indicates that metformin's most important effect in treating diabetes is to lower the hepatic production of glucose [32, 33].*

that hyperglycemia first detected during pregnancy be classified as either 'diabetes mellitus (DM) in pregnancy' or 'GDM' [36]. GDM is associated with short- and long-term sequelae on both, mother and offspring [37, 38].

During normal pregnancy, around the mid-pregnancy, a progressive insulin resistance develops that progresses during the third trimester. In early pregnancy, insulin secretion increases, while insulin sensitivity remain unchanged, decreased or increased whereas in mid pregnancy, insulin sensitivity declines progressively and worsens during the rest of the pregnancy, being worst in the late third trimester, which rebounds with the delivery of the placenta. Therefore, GDM usually develops in the late second trimester and disappears, instantly, post-delivery [39].

For GDM, lifestyle interventions such as daily exercise, medical nutrition therapy is the initial treatment while, metformin, the oral hypoglycemic agent is being considered as a substitute to insulin. The rationale behind using metformin in gestational and pre-existing diabetes during pregnancy is as metformin increases insulin sensitivity, reduces hepatic glucogeneogenesis and enhances peripheral glucose uptake, resulting in lowering of blood glucose with minimal risk of maternal hypoglycemia and weight gain [40].

Although, metformin has been shown to pass freely across the placenta [41], there are no reported adverse side effects to the fetus when it is used to treat women with infertility caused by poly-cystic ovary syndrome (PCOS) [42, 43]. Metformin is classified as a category B drug, which implies that there is no confirmation of animal or fetal toxicity or teratogenicity. The study of metformin in pregnancy revealed that the use of metformin in women with GDM was not associated with increased risk of congenital anomalies, or maternal and neonatal complications compared to insulin, except for higher rates of preterm labour [44].

Results of systematic review and meta-analysis had shown that metformin is better than insulin in reducing, maternal weight gain during pregnancy and the frequency of pregnancy induced hypertension, with no changes in the frequency of hypoglycemia and pre-eclampsia [45]. In addition, randomized controlled trials (RCT) suggest that metformin could be used to treat or

prevent pre-eclampsia [46]. Metformin is considered as the first-line drug in the management of type 2 diabetes due to its efficacy, tolerability and safety in non-pregnant individuals.

#### *1.1.3 Metformin in polycystic ovary syndrome*

Poly-cystic ovarian syndrome (PCOS) is a hormonal disorder often aggravated by obesity and insulin resistance. PCOS is an endocrine-metabolic dysfunction among 5–10% of women in reproductive age which is associated with metabolic disturbances that have a high impact in cardio metabolic diseases, such as insulin resistance [47–49].

PCOS is characterized by menstrual irregularities, low fertility, obesity and high blood levels of male hormones in reproductive aged women [50]. PCOS confirms insulin resistance which leads to the hypothesis of a pre-diabetic state with glucose intolerance, gestational diabetes mellitus and evident diabetes. Several studies show that insulin resistance stimulates the ovaries to produce male hormones, i.e., androgens. This causes stigmata of androgen excess such as hirsutism and acne. Metformin increases insulin sensitivity and decreases the production of ovarian androgen thereby normalizing the hormone levels, stabilizes menstrual irregularities and improves fertility and ovulation. It also directly inhibits the androgen production [51].

Metformin treats PCOS symptoms, such as irregular ovulation or menstrual cycles, and the excess of insulin in the body. It has also been made known to treat PCOS symptoms by reducing body mass index (BMI) and testosterone levels. Furthermore, metformin assists fertility and increases the chance of successful pregnancy and reduces the risk of early miscarriage, gestational diabetes, and inflammation associated with PCOS. Metformin is thus used as the drug of choice for the treatment of PCOS. More to that, metformin helps mothers carry their baby to full term [51, 52]. Metformin is strongly recommended in patients with metabolic syndrome and obesity [51].

#### *1.1.4 Metformin in obesity*

Obesity is a chronic disease accompanied with metabolic syndromes, such as diabetes, fatty liver diseases, and cardiovascular diseases (CVDs). Obesity is caused by an imbalance between energy intake and expenditure [53].

Metformin happens to be one of the drugs available for the treatment of obesity. Metformin acts on obesity by decreasing the appetite and reduced BMI levels. Metformin contains a primary anorectic factor which reduces the appetite. Leptin levels were found to be decreased on taking metformin. Moreover, glucagon like peptide-1 levels rise significantly on taking metformin. This promotes weight loss. It was observed that adults with severe obesity lost weight more significantly than mildly obese patients [54]. Metformin exerts its anti-obesity effects through increasing mitochondrial biogenesis, decreasing fatty acid uptake, and stimulating thermogenesis [55].

It acts by promoting sugar dysplasia restrains and reducing inhibition caused by insulin-induced expression of the glucose transporter protein, thus increasing glucose utilization [56]. Metformin is effective in reducing body weight and improving insulin sensitivity in adults, and is used to treat adolescents who are overweight or obese and unresponsive to changes in lifestyle or who present with insulin resistance [57]. Many studies support that metformin can promote weight loss in overweight or obesity patients [58, 59]. Based on the reports it is understood that clinical trials supports the efficacy and safety profiles of metformin in diabetes and weight gain prevention [60].

#### *1.1.5 Metformin in medication induced weight gain*

Studies have shown that use of antipsychotics increase the risk of weight gain, dyslipidemia and diabetes. Weight gain and abdominal adiposity which is directly associated with insulin resistance, dyslipidemia and risk of diabetes may be induced by second generation antipsychotics [61, 62]. Stimulation of appetite, reducing physical activity and impairing metabolic regulation is the mechanism of antipsychotics induced weight gain [63].

Metformin aids in weight loss. Drug induced weight gain can be reduced by metformin. It assists in reduction of weight for those who gain 10% of body weight than pre-treatment [63]. Metformin contains an anorectic factor and facilitates less hunger. This also aids in decreased appetite. Metformin causes decreased leptin levels, thus suppresses appetite. Metformin also increases the GLP-1 levels which enhances weight loss. Thus, metformin with lifestyle changes is effective in the treatment of weight gain induced by antipsychotics.

#### *1.1.6 Metformin in cancer*

New studies have shown that metformin is effective in killing cancer cells. In trials, people undergoing chemotherapy alone saw their cancer return, while for those on chemo and metformin, their tumors disappeared. Research has shown that those taking metformin are less likely to develop certain cancers. Metformin has been found to improve cancer prognosis as it inhibits cancer cell growth and proliferation. Evidence points that metformin inhibits growth, survival, and metastasis of different types of tumor cells, including those from breast, liver, bone, pancreas, endometrial, colorectal, kidney, and lung cancers [64].

Metformin prevented the growth and spreading of certain cancers in patients with type 2 diabetes. This proposed mechanism is through a known tumor-suppressant gene (LKB1), which activates AMPK. Metformin shows anticancer properties by direct and indirect regulation of cells' metabolism. The direct effects are mediated by AMPK dependent and -independent pathways. (i) Metformin activates AMPK, which leads to the inhibition of mTOR signaling, and thereby disturbs the protein synthesis, and suppresses the cell growth and proliferation [65]. As an antidiabetic drug, metformin decreases plasma glucose levels, thereby inhibiting cancer cell proliferation and survival [66].

Other studies reported that metformin could activate the immune response against cancer cells [67] or decrease NF-kB (nuclear factor-kB) activity, which results in a reduction in the secretion of pro-inflammatory cytokines [68].

Metformin activates AMPK and then induces p53 phosphorylation to prevent cell invasion and metastasis [69].

The different mechanisms antitumor action has been proposed which involves the following: (a) the activation of adenosine monophosphate kinase, (b) modulation of adenosine A1 receptor (ADORA), (c) reduction in insulin/insulin growth factors, and (d) inhibition of endogenous reactive oxygen species (ROS); and its resultant damage to deoxyribonucleic acid (DNA) molecule is another paramount antitumor mechanism [70].

Metformin reduces the proliferation of cancer cells and the possibility of malignancies in different types of cancer, including gastric carcinoma, pancreatic cancer, uterine cancer, medullary thyroid cancer [71]. **Figure 2** shows the mechanism of metformin in Cancer and **Figure 3** shows the direct and indirect effects of metformin in Cancer.

#### **Figure 2.**

*Mechanism of metformin in Cancer. The anticancer activity of metformin is associated with direct and indirect effects of the drug. The direct insulin-independent effects of metformin are mediated by activation of AMPK and a reduction in mTOR signaling and protein synthesis in cancer cells [72].*

#### **Figure 3.**

*Direct and indirect effects of metformin on cancer. Metformin activates AMPK leading to stabilization of TSC2 and inhibition of mTORC1 signaling and protein synthesis. Metformin can also directly target mTOR independently of AMPK and TSC2 [73].*

#### *1.1.6.1 Breast cancer*

Breast cancer (BC) is one of the most common malignancies occurring in females. Cellular glucose metabolism is linked tightly with the proliferation and development of breast cancer. Several studies suggested that metformin reduces

#### *Metformin: Pros and Cons DOI: http://dx.doi.org/10.5772/intechopen.99815*

the incidence of breast cancer in type 2 diabetes patients [74]. Cancer cells show enhanced glucose uptake and metabolism and prefer glycolysis. The noted specialty of metformin is to decrease glucose levels, thereby limiting the availability of energy for cancer cells. Metformin decreases FAS expression which is an essential component of the fatty acid synthesis pathway, therefore affecting the survival of cancer cells.

#### *1.1.6.2 Blood cancer*

Leukemia comprises 2.8% of all cancers and 3.4% of cancer-related deaths worldwide. The aberrant activation of the PI3K/AKT/mTOR pathway is one of the most common biochemical features of leukemia [75]. Metformin inhibits AKT/mTOR signaling, which is an effective approach to treat leukemia. Metformin plays a beneficial role in human lymphoma by inhibiting mTOR signaling without the involvement of AKT, and the suppression of mTOR subsequently leads to the suppression of growth of B cells and T cells [76].

#### *1.1.6.3 Colorectal cancer (CRC)*

CRC is also one of the most common cancers in the world. In 2004, relationship between metformin and CRC was demonstrated [77]. Metformin may exert its pharmacodynamic effects through the gut-brain-liver axis, but these mechanisms require further exploration. In the intestine, metformin increases glucose uptake and lactate concentrations. Administration of metformin increases the bile acid pool in the intestine that may affect GLP-1 secretion and cholesterol levels. In addition, metformin changes the microbiome, affecting the regulation of metabolism, such as glucose homeostasis, lipid metabolism, and energy metabolism [78]. These changes inhibits the development and progress of CRC.

#### *1.1.6.4 Bone cancers*

Compared with cancers initiating in bone tissue itself, invasion of metastatic cancers, especially breast, lung, and prostate cancers, into bones is more common [79]. All types of bone cancers influence the osteolytic process, and osteoblastic metastases occur through osteoclast activation or stimulant factors which are responsible for osteoblastic proliferation, differentiation, and formation [80].

#### *1.1.6.5 Endometrial cancer*

Metabolic syndrome like obesity and hyperglycemia is related to the development of endometrial cancer. Metformin is an effective anti-diabetic drug, studies have demonstrated the beneficial effect of metformin on endometrial cancer development by the mechanisms involving the mitochondrial OXPHOS suppression and AMPK activation which subsequently inhibit a variety of metabolic pathways, including STAT3, ZEB-1, ACC, mTOR, and IGF-1 [81].

#### *1.1.6.6 Melanoma*

Melanoma is the most aggressive skin cancer and is responsible for almost 80% of the skin cancer-related deaths. Due to its strong invasive ability, melanoma often metastasizes to the lymph nodes, liver, lungs, and even the central nervous system [82]. Metformin can induce cell cycle arrest in the G0–G1 phase in melanoma cells. Another study indicated that metformin can attenuate melanoma growth and

metastasis through inhibiting the expression of TRB3 (tribbles pseudokinase 3) in non-diabetic and diabetic mouse models [83]. Because of the activation effect of AMPK, metformin could influence melanoma cell death and proliferation and the tumor microenvironment. It will be interesting to investigate the effects of combination treatment of metformin with current therapies or other drugs to treat melanoma.

#### *1.1.7 Metformin in aging*

Aging is considered unavoidable and is modulated by genetic and dietary factors. The declining ability to regenerate damaged tissue and the deterioration in homeostatic processes are considered as biological features of aging [84]. Usually, the primary causes foraging are DNA damage and autophagy. Aging is a result of DNA damage, which can be induced by ROS, alkylation, hydrolysis, chemicals, and ultraviolet and other radiation [85]. Trials have shown metformin's efficacy in reducing the effects of aging, such as decreasing age-related illnesses, problems with cognitive function, and morbidity [86].

Metformin slows down aging and reduces the incidence of aging-related diseases such as neurodegenerative disease and cancer in humans. In spite of its widespread use, the mechanisms by which metformin exerts favorable effects on aging remain largely unknown [87]. The mechanisms by which metformin affects the aging process are partly dependent on the regulation of glucose metabolism. By inhibiting mitochondrial complex I, metformin reduces endogenous production of ROS and subsequently decreases DNA damage [88].

By activating AMPK, metformin is able to inhibit NF-kB signaling and attenuate cell inflammation [89]. Metformin also leads to decreased insulin levels, and suppresses IGF-1 signaling and mTOR signaling, resulting in suppression of inflammation and autophagy, which is beneficial to the aging process [90]. Besides, metformin was shown to have a function in the regulation of the microbiome, which may be another way to affect aging [91]. There are three main factors that are related with aging. They are oxidation, glycation and methylation. There is evidence that metformin acts as an anti-aging agent. It helps slow the rate of aging and retain youth characteristics for a longer period of time than compared to non-metformin users. There are ongoing research about the effect of metformin on anti-aging properties. Researches have proved that metformin is linked with anti-aging factors [92].

There are two mechanisms to describe aging. First one is ROS theory, i.e., reactive oxygen species [93]. The ROS theory explains that by products of oxidative phosphorylation are reactive oxygen species, i.e., free radicals. The free radicals increase significantly and damage other cells and organs. The ROS leads to DNA damage [64].

The second mechanism is TOR theory. Cellular pathway like IGF-1 axis, MAPK, AKT, PI3K stimulated by mitogens, growth factors, sugars and amino acids are said to inhibit aging. Caloric restriction suppresses the mTOR pathway. The activity of mTOR may be inhibited by rapamycin. Rapamycin has gero-suppressive effects. These include extending the lifespan, prevent age related disorders and reduce cost of patient care. AMPK activation led to an indirect inhibition of mTOR. Metformin acts as an AMPK activator [64, 94]. Metformin, Being an AMPK activator, metformin has been proved to have gero-suppressive effects. Extended longevity and lifespan were seen in those taking metformin. Autophagy plays a significant role in gero-suppressive mechanisms. Autophagy protects cell organelles and nutrient supply. Induction of autophagy extends the lifespan. Polyamines cause autophagy. Activation of autophagy induces processes associated with suppression of IGF and mTOR pathways. Therefore metformin acts as an activator of autophagy [95].

#### *1.1.8 Metformin in liver diseases*

Liver dysfunction may lead to many diseases, such as diabetes, non-alcoholic fatty liver disease, cirrhosis, non-alcoholic hepatitis, and hepatocellular carcinoma. Studies showed that metformin is safe in patients with cirrhosis. In diabetic patients, metformin caused a 50% reduction in hepatocellular carcinoma incidence and improved survival mainly by influencing cell growth and angiogenesis through the PI3K/AKT/mTOR signaling pathway [96]. In humans, metformin was also found to reduce the incidence of fatty liver diseases and to cause a histological response [97]. However, other studies showed that metformin failed to improve liver histology, hepatic steatosis, and inflammation [98].

#### *1.1.9 Metformin and cardiovascular diseases*

Hyperglycemia induces oxidative stress, resulting in lipoprotein dysfunction and endothelial dysfunction, increasing the risk of CVD. Metformin was shown to decrease the incidence of CVD in diabetes patients. Metformin was also shown to decrease irregular heartbeats and lower oxidative stress [86]. Through activating AMPK, metformin inhibits alpha-dicarbonyl-mediated modification of apolipoprotein residues, consequently ameliorating high density lipoprotein (HDL) dysfunction and reducing low density lipoprotein (LDL) modifications. Reductions in HDL dysfunction improve cholesterol transport and diminish the cardiovascular risk. Moreover, metformin improves endothelial oxidative stress levels and attenuates hyperglycemia-induced inflammation, decreasing the occurrence of CVD [99]. It has been shown that metformin improves the myocardial energy status through ameliorating cellular lipid and glucose metabolism via AMPK [100].

#### *1.1.10 Metformin and renal diseases*

Diabetes is considered as an important cause of renal diseases, and metformin is an interesting candidate to treat renal diseases, although its use was restricted previously [101]. Daily oral administration of metformin could improve kidney fibrosis and normalize kidney structure and function. These effects may be mediated by the AMPK signaling pathway, which can regulate cell growth and energy utilization. Another study found that in a CKD mouse model, metformin could suppress kidney injury and improve kidney function, through AMPK-mediated ACC signaling [102].

It is worth to note that appropriate dosage of metformin is very important in the treatment for renal diseases. The mechanisms underlying these kidney protective roles of metformin may be related to the regulation of glucose utilization, the decrease in cell inflammation, and oxidative stress [103]. The summary of metformin in different diseases and the underlying major mechanism is shown in the **Figure 4.**

#### **1.2 Cons (side effects) of metformin**

Metformin as all drugs, have unwanted effects which can be mild or serious side effects. The most common side effects are related to gut complications and include upset stomach, nausea, vomiting, diarrhea, light headedness, or a metallic taste in the mouth [104]. In general, older patients may be at an increased risk for some of its side effects, such as lactic acidosis or low blood sugar, due to other factors [104]. The minor side effects include gastrointestinal disturbances. The most common are anorexia, nausea, abdominal discomfort and diarrhea. Dose reduction or discontinuation of the drug may reduce or alleviate these symptoms. Out of the side effects,

**Figure 4.** *Summary of metformin in different diseases and the underlying major mechanism [103].*

lactic acidosis and vitamin B12 deficiency happens to be the major. Although rare, if lactic acidosis occurs, may be fatal, which may occur in presence of hypoxia and renal insufficiency [105].

#### *1.2.1 Lactic acidosis*

Lactic acidosis is a condition in which lactic acid builds up in the body, altering pH balance and potentially leading to complications [106]. Because metformin reduces the breakdown of lactate to glucose, the drug may induce lactic acidosis if it accumulates significantly. Metformin's exact mechanism of action in doing so is unknown. More frequently, the combination of this drug and an underlying health condition may trigger lactic acidosis [107].

The rate of developing lactic acidosis increases in patients with predisposing factors, such as renal impairment, hepatic disease, congestive heart failure or sepsis. Metformin is renally cleared. In cases of renal failure or decreased creatinine clearance, metformin accumulates. When this happens, it inhibits mitochondrial electron transport. Therefore, it increases anerobic metabolism and lactic production [108]. The levels of lactate increase in metformin taking patients. The pyruvate dehydrogenase inhibits conversion of lactate to glucose, thereby causes lactic acidosis [109].

Because metformin decreases liver uptake of lactate, any condition that may precipitate lactic acidosis is a contraindication. Patients with infections, recent surgery, kidney or liver damage, history of heart disease, respiratory failure, excessive alcohol consumption (due to depletion of NAD+ stores), or dehydration have an increased risk of lactic acidosis induced by metformin [110]. The FDA recommends avoiding the use of metformin in more severe chronic kidney disease, below the eGFR cutoff of 30 ml/minute/1.73 m<sup>2</sup> .

Lactate uptake by the liver is diminished with metformin use because lactate is a substrate for hepatic gluconeogenesis, a process that metformin inhibits. Metformin-associated lactate production may also take place in the large intestine, which could potentially contribute to lactic acidosis in those with risk factors. Elderly patients are also at risk for developing lactic acidosis [104, 111].

#### *1.2.2 Vitamin B12 deficiency*

A common report with long term metformin use is vitamin B12 malabsorption which leads to vitamin B12 deficiency [112, 113]. With increased metformin

#### *Metformin: Pros and Cons DOI: http://dx.doi.org/10.5772/intechopen.99815*

dosage, the incidence of vitamin B12 deficiency also increased [114]. In a study, it was proven that being treated with metformin had a 7% greater risk of vitamin B12 deficiency than with placebo [115].

The mechanisms leading to vitamin B12 deficiency may be explained by changes in small intestine motility. This cause increased bacterial growth and hence, consumption of vitamin B12. Metformin also inhibits the calcium dependent absorption of vitamin B12 [116]. Vitamin B12 is an essential nutrient for cognitive and cardiovascular function [117, 118]. Clinical manifestations of vitamin B12 deficiency include alteration in mental status, megaloblastic anemia and neurological damage [118].

#### *1.2.3 Hypoglycemia*

Metformin, itself, does not lead to a state of critically low blood sugar. In combination with other risk factors such as heavy alcohol drinking (or dehydration), the use of other drugs for diabetes, insufficient calorie intake, or bouts of heavy exercise, it may increase the chances of developing hypoglycemia [100]. Since metformin does not directly stimulate insulin secretion, hypoglycemia risk may be lower than for that of other oral anti-diabetes drugs. However, hypoglycemia in patients using metformin may occur in association with strenuous physical activity or fasting [119].

#### *1.2.4 Anemia*

Metformin can decrease the levels of vitamin B12 in our body. In rare cases, this can cause anemia or low levels of red blood cells. Metformin use is associated with early risk of anemia in individuals with type 2 diabetes. The mechanism for this early fall in hemoglobin is uncertain, but given the time course, is unlikely to be due to vitamin B12 deficiency alone [120].

Vitamin B12 (cobalamin) deficiency is a frequent cause of megaloblastic anemia that is evident through various symptoms [121]. However, the mechanism for these findings is unclear. Because development of anemia was not obviously associated with a rising mean cell volume (MCV) or macrocytosis, vitamin B12 deficiency is an unlikely explanation in most cases. We should evaluate anemia in metformin users as we would for any patient; if a thorough evaluation is unrevealing, we might cautiously attribute the anemia to metformin [120].

#### *1.2.5 Cognitive impairments*

A case–control study of over 7,000 patients with Alzheimer's disease showed that, compared to insulin treatments, sulfonylureas, and thiazolidinediones, metformin was associated with an increased incidence of Alzheimer's [122]. However, another study on approximately 1,500 people showed that the cognitive impairment associated with metformin may be alleviated with vitamin B12 and calcium supplements [123]. Controversies are seen as studies have reported that metformin was found to significantly reduce the occurrence of cognitive dysfunction in patients with T2D [124]. Several studies found that metformin improved cognitive abilities [125, 126]. The relationship between metformin and cognitive dysfunction in patients with T2D is controversial.

#### *1.2.6 Gastrointestinal*

Gastrointestinal upset is most common when metformin is first administered, or when the dose is increased. This can cause severe discomfort which can often be avoided by starting the drug at a low dose and increasing the dose gradually, but even with low doses, 5% of people may be unable to tolerate metformin. Long-term use of metformin has been associated with increased homocysteine levels and malabsorption of vitamin B12. Higher doses and prolonged use are associated with increased incidence of vitamin B12 deficiency.

#### **2. Conclusion**

Metformin, the drug initially approved and used for the treatment of Type 2 diabetes mellitus is proven to be effective in many other conditions such as gestational diabetes mellitus, obesity, hypersecretion of ovarian androgens, poly-cystic ovary syndrome (PCOS), anti-psychotic therapy induced weight gain, cancer treatment etc. There are ongoing research about the effect of metformin on anti-aging properties and proved that metformin is linked with anti-aging factors.

#### **Author details**

Shalini Sivadasan\*, Muthukumar Subramanian and Rajasekaran Aiyalu KMCH College of Pharmacy, Coimbatore, Tamil Nadu, India

\*Address all correspondence to: shaliniravichandran11@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## Interaction Studies of ACE Inhibitors with Antidiabetic Drugs

*Safila Naveed and Halima Sadia*

#### **Abstract**

Angiotensin converting enzyme (ACE)-inhibitors are effective in patients with mild to moderately severe hypertension, collagen vascular and cardiovascular disease. They are also used in the prevention and treatment of myocardial infarction and in the management of cardiac arrhythmias. Patients with cardiovascular diseases are generally on multiple medicines that's why it is imperative to study drug–drug interactions of medicines which are commonly taken together in any given case, as combined administration of different medicines can significantly influence the availability of drugs. In the present study we investigated the "*in vitro"* interactions of ACE inhibitors (enalapril, captopril and lisinopril) with frequently prescribed and co-administered drugs in simulated human body environments. These interactions were monitored by means of UV spectrophotometry and separation technique as RP-HPLC. Prior to start of actual drug interactions, the method of analysis of each drug was established and its various parameters validated for considering its use in testing of drug *in vitro* as well as in human serum. For this purpose, an attempt was made to develop a number of new HPLC methods for determination of ACE inhibitors (enalapril, captopril and lisinopril) and simultaneously with interacting drugs. These methods were optimized, validated and then successfully employed for the quantitation of enalapril, captopril and lisinopril and selected drugs in interactions studies. As a result, new methods for the quantitation of individual as well as multiple drugs were developed. The interacting drugs selected were antidiabetic drugs (metformin, glibenclamide, glimepride and pioglitazone. Interaction consequences revealed that the availability of enalapril was not affected in presence of antidiabetic drugss whereas the availability of captopril and lisinopril were altered in presence of NIDDMs.

**Keywords:** ACE Inhibitors, Antidiabetics, Interaction studies, HPLC, Method development

#### **1. Introduction**

#### **1.1 Angiotensin converting enzyme**

Angiotensin Converting Enzyme is an ectoenzyme and a glycoprotein with an appreciate molecular weight of 170,000 Do. Human angiotensin converting enzyme contains 277 aminoacid residues and has two homologous domains, each with a catalytic site and a region for binding Zn+2 [1, 2]. The degradation of bradykinin to inactive peptides occurs via action of ACE, thus ACE not only produces a potent vasoconstricton but also inactivates a potent vasodilator [3].

In 1965, Ferreira [4] studied the physiological effects of snake poisoning and discovered a specific component from the venom of the pit viper, *bothrops jararaca*, which inhibits degradation of the peptide bradykinin and potentiate hypotensive action of bradykinin. These factors originally designated as bradykinin potentiating factors (BPFs), were isolated and found to be a family of peptides containing 5–13 amino acid residues. Bakhle [5] reported that these same peptides had an inhibitory activity on ACE of dog lung homogenate and inhibited the enzymatic conversion of angiotensin I to angiotensin II. Hans Brunner and John Laragh [6] administered it to hypertensive patients and showed that it was extremely effective in lowering blood pressure. The structural requirements for substrates of angiotensin converting enzyme to cleave a substrate are found similar to those observed with carboxypeptidase A of bovine pancrease [7, 8]. The substrate specificity and other properties of angiotensin converting enzyme suggested that it was a zinc metallopeptidase, similar in mechanism to carboxypeptidase A, an enzyme whose active site had been well characterized by x-ray crystallography and other methods [9]. In 1970, Ferreira and Greene [10] isolated and characterized the first peptide, a bradykinin-potentiating pentapeptide that they called BPP5a; it also inhibited ACE and transiently lowered blood pressure in animal models. The significance of ACE in the pathogenesis of hypertension was not fully appreciated until 1977's, when Ondetti [11] first isolated and then synthesized the naturally occurring nonpeptide, teprotide. He proposed a hypothetical model of the active site of ACE and used it to predict and design compounds that would occupy the carboxy- terminal binding site of the enzyme [12]. Cushman and Ondetti first created succinyl-L-proline, which showed slight positive activity. Inhibitory activity increased 15 to 20 times when they substituted a methyl group in the 2 position of succinyl group. Finally to enhance the binding capacity of substrate structure and zinc of the enzyme they replaced succinyl COOH with sulfhydryl, a 2000 times increase in inhibitory potency was achieved. ACE inhibitors entered the antihypertensive drug market during the 1980. Manolio [13] explored new types of drugs in preventing cardiovascular mortality. Captopril, a specific potent inhibitor of ACE, showed excellent anti-hypertensive properties in clinical trials and had a major impact on the treatment of cardiovascular disease [14].

#### *1.1.1 Chemistry*

The most thoroughly studied of the peptide inhibitors of converting enzyme is the nonapeptide known as teprotide, having the structure, Pyoglu-Tro-Arg-Pro-Glnlle-Pro-Pro.Teprotide acts as a competitive inhibitor of converting enzyme, with an affinity for the enzyme much higher than that of angiotensin I. It is not itself a substrate for the enzyme. Although converting enzyme will cleave many different C-terminal dipeptide residues, it will not cleave peptides with proline in the penultimate position. As noted, the penultimate proline in angiotensin II, indeed, is responsible for its refractoriness to further cleavage by converting enzyme. Moreover, the presence of Pyro Glu at the N-terminus renders teprotide refractory to amino peptidases; this confers further stability and effectiveness in vivo. Nevertheless, teprotide has a relatively short duration of action and must be given parentally to be effective [11]. The optimum pH of angiotensin converting enzyme was found to vary with the substrate employed and to be influenced by the presence or absence of chloride ion. With longer peptide substrates such as angiotensin I or bradykinin in the presence of chloride ion, the optimal pH for hydrolytic action of the converting enzyme was about 7.5; with tripeptide substrates such as Z-Phe-His-Leu, Hip-His-Leu, or Hip-Gly-Gly, it was about pH 8.5 [15, 16]. Studies of the hydrolysis of synthetic substrate of ACE [17, 18] and hippuryl di and tripeptides [19] shows that enzyme tolerate changes at antepenultimate position of a peptide

#### *Interaction Studies of ACE Inhibitors with Antidiabetic Drugs DOI: http://dx.doi.org/10.5772/intechopen.99795*

substrate especially aromatic amino acids such as phenylalanine which contributes greatly to the overall affinity for the enzyme. A tripeptide with an acylated terminal amino group is the simplest peptide cleaved by the enzyme. However, the tripeptide Z-Phe-His-Leu, analogous to the terminal tripeptide sequence of angiotensin I, binds to the active site of angiotensin converting enzyme as well as the intact decapeptide. Peptides such as angiotensin II with a penultimate proline residue [20]. The orally effective ACE-inhibitor was developed by a rational approach that involved analysis of the inhibitory action of teprotide, inferences about the action of converting enzyme on its substrates, and analogy with carboxy peptidase A, which was known to be inhibited by d-benzylsuccinic acid. Ondetti and Cushman urged that inhibition of converting enzyme might be produced by succinyl amino acids that corresponded in length to the dipeptide cleaved by converting enzyme. This proved to be true and led ultimately to the synthesis of a series of carboxy or mercapto alkanoyl derivatives that acted as competitive inhibitors of the enzyme [21].

#### *1.1.2 Mechanism of action*

These drugs block the angiotensin converting enzyme that cleaves the terminal two peptides from angiotensin I (decapeptide) to form the potent vasoconstrictor angiotensin II (octapeptide) [22, 23] and lower the BP by reducing peripheral vascular resistance without reflexly increasing cardiac out put rate, and contractility [22]. They also inhibit the rate of bradykinin inactivation thus resulting in vasodilation, they also decrease the secretion of aldisterone resulting in decrease of sodium and water retention.

#### *1.1.3 Pharmacokinetics*

ACE-inhibitors are given by mouth, the oral bioavailability of this class of drugs ranges from 13–95% [24, 25]. Most of the ACE inhibitors are administered as prodrugs that remain inactive until esterified in the liver [26]. Fosinoprilate is excreted via biliary duct, elimination of the diacid is polyphasic and there is a prolong terminal elimination phase, which is considered to represent binding to ACE at saturate binding site. This bond fraction does not contribute to accumulation of drug following multiple doses [27, 28].

#### *1.1.4 Therapeutic use*

ACE-inhibitors are effective in patients with mild to moderately severe hypertension, with normal or low plasma renin activity, with collagen vascular disease, with cardiovascular and in anephric disease [29–36]. They cause a reduction in left ventricular hypertrophy, and in plasma fibrinogen level [37, 38]. They are also used in the prevention and treatment of myocardial infarction [39, 40], and in the management of cardiac arrhythmias [41, 42]. They can decrease the progression of atherosclerosis [43], microalbuminuria [44] and diabetic retinopathy [45–47] and produce beneficial effect in Bartter's syndrome [48].

#### *1.1.5 Adverse effects*

Pronounced hypertension may occur at the start of therapy with ACE-inhibitors particularly in patients with heart failure, and in sodium or volume depletion patients [49–51]. They cause hyperkalemia in patients with renal insufficiency or in patients taking k + −sparing diuretic, k + −supplement, beta blockers or NSAID's [23, 52] and produce cough in hypertensive patient [53, 54]. Altered liver function,

cholestatic jaundice, hepatitis, hepatotoxicity [55] and aplastic anemia [56] have also been reported. They can produce a complex and contradictory effect on kidney and induce renal insufficiency in patients having bilateral renal artery stenosis, heart failure or diarrhea [57–61]. Angioedema is a rare but potentially life-threatening side effect of ACE inhibitors [62–68] can cause a number of fetal anomalies [69, 70]. Scalded mouth syndrome [71] and drug induced pulmonary-infiltration with eosinophilia syndrome (PIE-syndrome) is a rare complication [72]. With use of ACE inhibitors, anaphylactoid reactions are also reported [73, 74].

#### *1.1.6 Contraindications*

Experimental and clinical data conclude that use of ACE inhibitors should be avoided in all trimester of pregnancy [75, 76]. Patients with peripheral vascular disease are at high risk of renal failure with this therapy [77] also contraindicated in known hypersensitivity to any ACE inhibitors [78].

#### *1.1.7 Overdosage*

There have been reports of over dosages with captopril and enalepril [79–81], the main effect is hypotension [82, 83] which usually responds to supportive treatment and volume expansion, pressor agents are rarely required. Infusion of angiotensin amide may be considered if hypotension persists [84, 85].

#### *1.1.8 Drug interactions*

Hypotensive effect of ACE inhibitors decreased when given in combination with non-steroidal anti-inflammatory drugs [86] but this effect is enhanced with calcium-channel blockers [87] and beta-blockers [88]. Granulocytopenia occurs after combine therapy of ACE inhibitors and interferones [89], the nitritoid reaction occurs with concomitant use of gold salt and ACE inhibitors [90]. Cytokines antagonize the hypotensive effect of ACE inhibitors [91], severe hypokalaemia occurs with potassium depleting diuretics [92] and potassium-sparing diuretics produced hyperkalaemia [93–95]. ACE inhibitors could increase potassium levels in the body [96, 97]. Alpha-blockers enhance hypotensive effect of ACE inhibitors [98]. Iron supplementation successfully decreases cough induced by ACE-inhibitors [99] and can interfere with the absorption of ACE inhibitors [100]. Hypoglycemic effect is enhanced with antidiabetics and insulin [101, 102]. Azathioprine and ACE inhibitors combination is associated with anemia [103]. Marked hypotension occurs in patients receiving general anesthetics and ACE inhibitors [104]. The risk of bone marrow depression is increased in patients taking concomitant therapy of ACE-inhibitors and immunosuppressive agents [76]. **Table 1** shows some example of ACE Inhibitors.

#### **1.2 Antidiabetic drugs**

Type II or non insulin dependent diabetes mellitus (NIDDM) formerly known as maturity-onset or adult-onset diabetes. Approximately 95% of patients are being affected by the type II form [105, 106]. NIDDM are being increasingly diagnosed as its importance as a risk factor for the development of cardiovascular disease and many drugs has been known to interfere with glucose control. The greatest effect was seen with propranolol and the least with cardioselective and less lipophilic beta-blockers, nifedipine has been associated with deterioration in glucose control but verapamil has been found to have a beneficial effect on glucose control. Antihypertensive drug clonidine has not been shown to result in deterioration in

glucose control when used in NIDDM. Long term therapy with the more specific agonist guanfacine was reported to have a beneficial effect on glucose tolerance [107]. **Table 2** shows, examples of antidiabetic drugs.


#### **Table 1.**

*Examples of ACE inhibitors.*


**Table 2.** *Examples of anti-diabetic.*

#### **2. Experimental**

#### **2.1 Materials**

Raw materials used were of pharmaceutical purity and were obtained from different Pharmaceutical Companies (**Table 3**). Tablets were purchased from local


#### **Table 3.**

*Drugs, brands and manufacturers.*

pharmacy and each product was labeled and expiry date not earlier than two years, at the time of these studies were noted.

#### *2.1.1 Reagents*

Analytical grade reagents were used during the whole experimental procedures. Methanol and acetonitrile were of (HPLC grade) (TEDIA®, USA). Other reagents include hydrochloric acid, sodium hydroxide, sodium chloride, potassium dihydrogen orthophosphate, disodium hydrogen orthophosphate, ammonium chloride, 10% NH3 solution, phosphoric acid 85% (Merk, Germany). Organic solvents used were methanol, ethanol, ethyl acetate, chloroform, acetronitrile, triethylamine and DMSO (Merck Grade).

#### *2.1.2 Equipments*

UV visible spectrophotometer (Model 1601, Shimadzu, Japan) with 10-mm path length connected to a P-IV computer loaded with Shimadzu UVPC version 3.9 software was used in these studies. Deionizer, Stedec CSW-300 used for deionization of water. The dissolution equipment was the B.P. 2009 standards. Chromatographic studies were carried out by using two Shimadzu HPLC systems, one equipped with LC-10 AT VP pump, SPD-10 A VP UV–*vis* detector and other HPLC system was equipped with LC-20AT and SPD-20A UV/VIS detector utilizing Hypersil, ODS, C18 (150 × 4.6 mm, 5micron) and Purospher® STAR RP-18 column. Chromatographic data were recorded using a CBM-102 Shimadzu. Shimadzu Class-GC 10 software (version 2) for data acquisition and mathematical calculations.

IR studies were carried out by FTIR Prestige-21 spectrophotometer Shimadzu. Spectral treatment was performed using Shimadzu IRsolution 1.2 software. The H1 -NMR spectra were recorded on a Bruker AMX 500 MHz spectrometer using TMS as an internal standard. Melting points were recorded by Gallenkamp melting point apparatus.

#### **2.2 Methods**

#### *2.2.1 Preparation of simulated gastric juice and buffers*

0.1 N hydrochloric acid was prepared by diluting 9 mL hydrochloric acid of analytical grade (11 N) in a liter volumetric flask and the volume was made up to the mark with de-ionized water. Chloride buffer of pH 4 was prepared by dissolving 3.725 g of potassium chloride in deionized water in one liter and 0.1 N HCl was used for pH adjustment. For preparation of phosphate buffer of pH 7.4, 0.6 gm of potassium dihydrogen orthophosphate, 6.4 g of disodium hydrogen orthophosphate and 5.85 g of sodium chloride were dissolved in sufficient deionized water to produce 1000 mL and the pH adjusted. For preparation of ammonia buffer of pH 9, 4.98 g of ammonium chloride was dissolved in 1000 mL of deionized water and pH adjusted with 10% ammonia.

### *2.2.2 Construction of the calibration curve of drugs*

The above prepared working standard solutions of all drugs were scanned in the region 200–700 nm against the reagent blank and absorbance maxima was recorded as shown in **Table 4**. Calibration curves were constructed between concentration and absorbance. Epsilon values and linear coefficients were calculated in each case at all above described pH values. Beer Lambert's law was obeyed at all concentrations and pH.

#### *2.2.3 Monitoring of drug interactions of enalapril, captopril and lisinopril by high performance liquid chromatography*

HPLC methods for simultaneous determination of enalapril, captopril and lisinopril with NSAIDs, H2-receptor antagonist, statins, antidiabetic drugs, metals and antacids in raw materials, pharmaceutical dosage forms or in human serum are developed and validated according to ICH guidelines. These methods were then applied to drug–drug, drug metals and drug antacid interaction studies.

#### *2.2.4 Chromatographic conditions*

The isocratic elution was performed at ambient temperature with two different types of columns. Hypersil, ODS, C18 (150 × 4.6 mm, 5micron) and Purospher® STAR RP-18, for assay of enalapril, captopril and lisinopril and simultaneous determination of these drugs with interacting drugs respectively. The mobile phase, flow rate, wavelength UV detection were varied as cited in **Table 5**. Sample volume of 20 μL was injected in triplicate onto the HPLC column and elute was monitored at different wavelengths.

#### *2.2.5 Preparation of standard solutions*

Stock reference standard solutions of all drugs were prepared daily by dissolving appropriate amounts of each drug in mobile phase to yield final concentrations


**Table 4.** *Absorbance maxima.*


**Table 5.**

*Chromatographie conditions of HPLC methods.*

300 μg mL−1. For the calibration standards, calibrators of each drug were prepared by making serial dilutions from stock solutions. All solutions were filtered through 0.45 μm filter and degassed using sonicator.

#### *2.2.6 Preparation of pharmaceutical dosage form samples*

Pharmaceutical formulations of the respective brands, commercially available in Pakistan were evaluated. In each case, groups of twenty tablets were individually weighed and finely powdered in a mortar. Weighed portion of the powder equivalent to the suitable amount of drug (according to the labeled claimed) was transferred into a 100 mL volumetric flask completely dissolved in mobile phase and then diluted with this solvent up to the mark, a portion of this solution was filtered through a disposable 0.45 μm filter and then injected.

#### *2.2.7 Preparation of standard drug plasma solutions*

Blood samples were collected from healthy volunteers and then centrifuged at 3000 rpm for 10 minutes and supernatant was stored at −20°C. After thawing, serum was deprotinated by acetonitrile and spiked daily with working solutions to produce desired concentrations of enalapril and interacting drugs. 10 μL volume of each sample was injected and chromatographed under above conditions.

#### **2.3 Method development and optimization**

HPLC methods were developed and optimized for certain parameters before method validation. The optimization of the analytical procedure has been carried out by varying the mobile phase composition, flow rate, pH of the mobile phase, diluents of solutions and wavelength of analytes in order to achieve symmetrical peaks with good resolution at reasonable retention time.

#### *2.3.1 Method validation*

All validation steps were carried out according to the ICH guidelines such as system suitability, selectivity, specificity, linearity (concentration–detector response relationship), accuracy, precision and sensitivity i.e. detection and quantification limit.

#### *2.3.2 System suitability*

System suitability of the method was evaluated by analyzing five replicate analyses of the drug at a specific concentration for repeatability, peaks symmetry (symmetry factor), theoretical plates of the column, resolution between the peaks of enalapril and other drugs, mass distribution ratio (capacity factor) and relative retention.

#### *2.3.3 Specificity and linearity*

The drugs were spiked with pharmaceutical formulations containing different excepients. The linearity of the method was evaluated at different concentrations with different groups. Linear correlation coefficient, intercept and slope values were calculated for statistical analysis.

#### *2.3.4 Accuracy and precision*

The accuracy of the method was calculated at three concentration levels (80, 100 and 120%) by spiking known quantities of the drug analytes. Three injections of each solution were injected to HPLC system and % recovery was calculated in each case.

For the precision of the method, six replicates of each level were injected to system on two different non-consecutive days in each case and %RSD was calculated.

#### *2.3.5 Limit of detection and quantification*

Detection limit (LOD) of the method was calculated by the formula LOD = 3.3 SD/slope. The quantitation limit (LOQ ) is the lowest level of analyte that is accurately measured and it was evaluated as ten times the noise level LOQ =10ơ/S; where ơ is the standard deviation of the lowest standard concentration and S is the slope of the standard curve.

#### *2.3.6 Robustness*

Robustness was performed by making minor changes in the percentage of mobile phase (methanol, water and acetonitrile) wave length, pH and flow rate. Therefore, five repeated samples were injected under small variations of each parameter. When a parameter was changed ±0.2% (in flow rate), ± 0.2% pH and ± 5% wave length from its optimum condition.

#### *2.3.7 Ruggedness*

Ruggedness of our method was determined in two different labs. Lab 1 was the Research Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi while other lab was lab 9, Department of Chemistry, Faculty of Science, University of Karachi. Two different instruments one was LC 10 and LC 20. Two different columns Purospher STAR C18 and Hypersil ODS were used.

#### *2.3.8 Interaction studies by HPLC*

Enalapril solution was mixed with each solution of interacting drug separately that gave the final concentration of 100μgmL−1 for each constituent. These were kept in water bath maintained at 37°C for 3 hours. An aliquot of 5 mL was withdrawn after every 30 minutes intervals, after making appropriate dilutions was filtered through 0.45 μ filter paper and three replicates were injected to HPLC system. The concentration of each drug was determined and % recovery was calculated and the same procedure was applied for captopril and lisinopril.

#### **3. Result and discussion**

#### **3.1 Simultaneous quantitation of enalapril and antidiabetic drugs (metformin, glibenclamide and glimepiride)**

There are number of HPLC methods reported for the quantitation of metformin using UV detector [108, 109] liquid chromatography–tandem mass spectrometry [110] and from human plasma [111]. Moreover, there are many methods reported for the simultaneous analysis of metformin with other antidiabetics [112, 113]. Likewise, there are methods reported for the analysis of glibenclamide from pharmaceutical formulations [114], human plasma [115, 116] using HPLC. Similarly, there are methods reported for the simultaneous analysis of glibenclamide with other anti-diabetics. However, no method reported in the literature for the simultaneous quantitation of enalapril, metformin, glibenclamide and glimepride.

#### *3.1.1 Method optimization and chromatographic conditions*

In the present investigation the best separation of enalapril and antidiabetic drugs was achieved using a Hypersil, ODS, C18 (150 × 4.6 mm, 5micron) column which provides efficient and reproducible separation of the components. Using other type of column under similar experimental condition, the separation lasted about 11 minutes. A mobile phase of methanol: water (70:30 v/v) having pH adjusted with phosphoric acid to 2.8 provided a reproducible, baseline resolved peak. Small changes in pH of the mobile phase had a great influence to the chromatographic behavior of these drugs, higher pH of the mobile phase also results in peak tailing and at a lower pH retention time of antidiabetic drugs and enalapril was delayed. It is obvious from the chromatogram (**Figure 1**) that antidiabetic drugs and enalapril eluted out forming symmetrical peaks and were well separated from each other. The method was found to be rapid as the drugs separated in a very short time i.e. enalapril 3.6 min and metformin, glibenclamide and glimepiride elution time was 2.4, 8.5 and 10.9 min respectively, which is important for routine analysis. The advantages of this method are ease of operation, short analysis time (total run time < 12 minutes), utilization of readily available cost-effective solvents, no matrix interferences, and satisfactory limit of quantification to enable pharmacokinetic studies of enalapril and NIDDMs.

#### *3.1.2 Method validation*

The developed method was validated by ICH guidelines [117]. It includes various parameters for example system suitability, selectivity, specificity, linearity, accuracy test, precision, robustness, ruggedness, sensitivity, limit of detection and quantification.

#### *3.1.2.1 System suitability*

The HPLC system was equilibrated with the initial mobile phase composition, followed by 6 injections of the same standard to evaluate the system suitability on each day of method validation. Parameters of system suitability are peaks symmetry (symmetry factor), theoretical plates of the column, resolution, mass distribution ratio (capacity factor) and relative retention as summarized in **Table 6**.

*Interaction Studies of ACE Inhibitors with Antidiabetic Drugs DOI: http://dx.doi.org/10.5772/intechopen.99795*

**Figure 1.** *A representative chromatogram of, and (a) MET (b) ENP (c) GLB (d) GMP in formulation and serum.*


**Table 6.**

*System suitability parameters.*

#### *3.1.2.2 Linearity*

Linearity is generally reported as the variance of the slope of the regression line. Linearity was tested with known concentrations of ENP, MET, GLB and GMP i.e. 2.5, 5, 10, 25, 50 and 100 μgmL−1 respectively. Injected concentrations versus area were plotted and the correlation coefficients were calculated which are shown in **Table 7**.


**Table 7.**

*Regresssion statistics LOD and LOQ.*

#### *3.1.2.3 Accuracy*

Method accuracy was evaluated as the percentage of recovery by estimation of all investigated analytes in presence of various commonly used tablets' excepients at three levels of concentrations that were 80, 100 and 120%. Each sample was injected five times and accuracy was determined in range of 98.6–102.3% (**Table 8**). No significant difference observed between amounts added and recovered without serum and with serum. Thus, used excepients did not interfere with active present in tablets.

#### *3.1.2.4 Precision*

Precision was evaluated by carrying out six independent sample preparation of a single lot of formulation. The sample solution was prepared in the same manner as described in sample preparation. Percentage relative standard deviation (%RSD) was found to be less than 2% for within a day and day to day variations, which proves that method is precise. Results are shown in **Table 9**.

#### *3.1.2.5 Sensitivity*

The limit of quantitation (LOQ ) of the method as signal/noise of ENP, MET, GLB and GMP were found to be 4.6, 0.96, 0.58 and 0.32 μgmL−1 respectively.


#### **Table 8.**

*Accuracy of ENP and NIDDM drugs.*


#### *Interaction Studies of ACE Inhibitors with Antidiabetic Drugs DOI: http://dx.doi.org/10.5772/intechopen.99795*

#### **Table 9.**

*Inter day and intraday precision of ENP and NIDDM drugs.*

Similarly a signal/noise of 3, a LOD of ENP, MET, GLB and GMP were determined to be 1.53, 0.317, 0.19, and 0.1 μgmL−1 respectively.

#### *3.1.2.6 Ruggedness*

The ruggedness of this method was calculated in two different labs with two different instruments. The method did not show any notable deviations in results from acceptable limits.

#### *3.1.2.7 Robustness of method*

To evaluate the robustness of the developed RP-HPLC method, small deliberate variations in the optimized method parameters were done. The effect of change in flow rate, pH and mobile phase ratio on the retention time and tailing factor were studied. The method was found to be unaffected by small changes like ±0.1 change in pH, ± 0.1 change in flow rate and ± 1 change in mobile phase.

#### **3.2 Simultaneous determination of captopril and antidiabetic drugs (metformin, pioglitazone and glibenclamide)**

The aim of the present study was to establish an efficient, reliable, accurate, precise and sensitive method for the separation and quantitative determination of both drugs simultaneously. These drugs belonged to different classes that could be co-administrated in a number of cases. Simultaneous determination of these drugs is desirable as this would allow more efficient generation of clinical data and could be performed at more modest cost than separate assays. We have developed the method for the simultaneous determination of captopril, metformin, pioglitazone and glibenclamide. The method has been validated according to ICH guidelines and was found to be reproducible. Further, this validated method was used to study the possible *in vitro* interactions of captopril with (metformin, pioglitazone and glibenclamide). Several problems were resolved in the simultaneous determination of compounds investigated.

#### *3.2.1 Method optimization and chromatographic conditions*

To optimize the operating conditions for isocratic RP-LC detection of all analytes, a number of parameters such as the mobile phase composition, pH and the flow rate were varied. Various ratios (50:50, 60:40, 70:30 v/v) of methanol: water were tested as starting solvent for system suitability study. The variation in the mobile phase leads to considerable changes in the chromatographic parameters, like peak symmetry, capacity factor and retention time. The pH effect showed that optimized conditions are reached when the pH value is 2.8, producing well resolved and sharp peaks for all drugs assayed. However, the ratio of (70:30 v/v) methanol: water pH adjusted to 2.8 with phosphoric acid as mobile phase (filtered through a 0.45 micron filter), a flow rate of 1.0 mLmin−1 using wavelength 230 nm was chosen as optimal condition. Retention time for captopril was found to be 3.3 minute, metformin, pioglitazone and glibenclamide 2.4, 2.8, 7.2 minutes respectively (**Figure 2**).

#### *3.2.2 Method validation*

The developed method was validated by ICH guidelines [5]. It includes various parameters for example system suitability, selectivity, specificity, linearity, accuracy test, precision, robustness, ruggedness, sensitivity, limit of detection and quantification (**Table 10**).

#### *3.2.2.1 Linearity*

Linearity was studied by preparing standard solutions at different concentration levels. The linearity range for CAP and antidiabetics was found to be 2.5–100 μgmL−1 and 0.625–25 μgmL−1, respectively, regression equations for CAP and antidiabetics are given in **Table 11**.

#### *3.2.2.2 Accuracy*

Method accuracy was evaluated as the percentage of recovery by estimation of all investigated analytes in presence of various commonly used tablets' excepients at three levels of concentrations that were 80, 100 and 120%. Each sample was injected five times and accuracy was determined in range of 98.45–102.2%. No significant difference was observed between amounts added and recovered without serum and with serum (**Table 12**).

*Interaction Studies of ACE Inhibitors with Antidiabetic Drugs DOI: http://dx.doi.org/10.5772/intechopen.99795*

#### **Figure 2.**

*A representative chromatogram of (a) metformin (b) pioglitazone (c) captopril and (d) glibenclamide in formulation.*

#### *3.2.2.3 Precision*

Precision was evaluated by carrying out six independent sample preparations of a single lot of formulation. The sample solution was prepared in the same manner as described in sample preparation. Percentage relative standard deviation (%RSD) was found to be less than 2% for within a day and day to day variations, which proves that method is precise (**Table 13**).

#### *3.2.2.4 Sensitivity*

The limit of quantitation (LOQ ) of the method as signal/noise of CAP, MET, PGL and GLB were found to be 2.3, 1.5, 2.3and 2.3 μgmL−1 respectively. Similarly a signal/noise of 3, a LOD of CAP, MET, PGL and GLB were determined to be 0.7, 0.4, 0.7, and 0.7 μgmL−1, respectively.

#### *Metformin - Pharmacology and Drug Interactions*


#### **Table 10.**

*System suitability parameters.*


#### **Table 11.**

*Regression characteristics.*


#### **Table 12.**

*Accuracy of captopril and antidiabetic drugs.*

#### *3.2.2.5 Ruggedness*

Ruggedness of this method was evaluated in two different labs with two different instruments. The method did not show any notable deviations in results from acceptable limits.


#### *Interaction Studies of ACE Inhibitors with Antidiabetic Drugs DOI: http://dx.doi.org/10.5772/intechopen.99795*

#### **Table 13.**

*Inter day and intraday precision of captopril and NIDDM drugs.*

#### *3.2.2.6 Robustness of method*

To evaluate the robustness of the developed RP-HPLC method, small deliberate variations in the optimized method parameters were done. The effect of change in flow rate, pH and mobile phase ratio on the retention time and tailing factor were studied. The method was found to be unaffected by small changes like ±0.1 change in pH, ± 0.1 change in flow rate and ± 1 change in mobile phase.

#### **3.3 Simultaneous determinations of lisinopril, pioglitazone, glibenclamide and glimepiride**

There is no method reported for the simultaneous determination of LSP and antidiabetic drugs using HPLC however there are methods for the determination of lisinopril [118, 119], similarly, there are methods reported for the simultaneous analysis of anti-diabetics. An isocratic reversed phase high-performance liquid

chromatographic (RP-HPLC) method has been developed for the simultaneous determination of lisinopril and antidiabetic drugs pioglitazone, glibenclamide and glimepride in bulk, dosage formulations and human serum and used for interaction studies.

#### *3.3.1 Method optimization and chromatographic conditions*

To develop a precise, accurate and suitable RP- HPLC method for the simultaneous estimation of LSP with antidiabetic drugs, different mobile phases were tried and the proposed chromatographic conditions were found to be appropriate for the quantitative determination. The short analysis time (<8 min) also enables its application in routine and quality-control analysis of finished products. pH of mobile phase containing methanol: water (80:20),was adjusted to 2.9 with phosphoric acid.The mobile phase was filtered on a 0.45 micron filter and then sonicated for 10 min. The flow rate was set to 1.0 mLmin−1. The retention time for LSP was found to be 2.0 minute pioglitazone 2.6 minute, for glibenclamide was 5.3 minute and glimepride 6.1 minute.

#### *3.3.2 Method validation*

The developed method was validated by ICH guidelines, it includes system suitability, selectivity, specificity, linearity, accuracy test, precision, robustness, ruggedness, sensitivity, limit of detection and quantification.

#### *3.3.2.1 System suitability*

The HPLC system was equilibrated initially with the mobile phase, followed by 6 injections of the same standard to evaluate the system suitability on each day of method validation. Parameters of system suitability are peaks symmetry (symmetry factor), theoretical plates of The column, resolution, mass distribution ratio (capacity factor) and relative retention as summarized in **Table 14**.

#### *3.3.2.2 Linearity*

Linearity was studied by preparing standard solutions at different concentration levels. The linearity range for LSP, PGL, GLB and GMP was found to be 2.5–100 μgmL−1. The regression equation for LSP and antidiabetic drugs were given in **Table 15**.

#### *3.3.2.3 Accuracy*

The accuracy of the method was evaluated as the percent recovery by estimation of all investigated analytes in presence of various commonly used tablets' excepients at three levels of concentrations that were 80, 100 and 120%. Each sample was injected five times and accuracy was determined in range of 98.45–102.2%. No significant difference observed between amounts added and recovered without serum and with serum (**Table 16**). Thus, used excepients did not interfere with active present in tablets (**Figure 3**).

#### *3.3.2.4 Ruggedness*

Ruggedness of the method was calculated in two different labs with two different instruments. The method did not show any notable deviations from acceptable limits.

#### *Interaction Studies of ACE Inhibitors with Antidiabetic Drugs DOI: http://dx.doi.org/10.5772/intechopen.99795*


#### **Table 14.**

*System suitability parameters.*


#### **Table 15.**

*Regression statistics LOD and LOQ.*


#### **Table 16.**

*Accuracy of LSP and NIDDM drugs.*

#### *3.3.2.5 Precision*

Precision was evaluated by carrying out six independent sample preparation of a single lot of formulation. The sample solution was prepared in the same manner as described earlier. Relative standard deviation was found to be less than 2% for within a day and day to day variations, which proves that method is precise (**Table 17**).

#### **Figure 3.**

*A representative chromatogram of (1) lisinopril (2) pioglitazone (3) glibenclamide and (4) glimepride in formulation and serum.*


#### **Table 17.**

*Inter day and intraday precision of LSP and N1DDMdrugs.*

#### *3.3.2.6 Sensitivity*

Limits of quantitation of the method as signal/noise of 10, for lisinopril, pioglitazone, glibenclamide and glimepride were found to be 1.6, 0.23, 0.29 and 0.12 μgmL−1respectively. Similarly a signal/noise of 3, LOD of lisinopril, pioglitazone glibenclamide and glimepiride were determined to be 0.53, 0.07, 0.09 and 0.04 μgmL−1.

#### *3.3.2.7 Robustness of method*

To evaluate the robustness of the developed RP-HPLC method, small deliberate variations in the optimized method parameters were done. The effect of change in flow rate, pH and mobile phase ratio on the retention time and tailing factor were studied. The method was found to be unaffected by small changes like ±0.1 change in pH, ± 0.1 change in flow rate and ± 1 change in mobile phase.

#### **3.4 Interaction of ACE inhibitors with antidiabetic drugs**

Hypertension in diabetics represents an important health problem as the combination of these diseases is common, carries significant morbidity and mortality and is frequently difficult to treat. The prevalence of hypertension in diabetic people is probably 1.5–2 times higher than in the general population [118]. Reduction of cardiovascular risk is therefore a high priority in the management of diabetes. Micro albuminuria is an important predictor of cardiovascular events and forms one of the components of insulin resistance/metabolic syndrome, which confers a particularly high risk of cardiovascular death [119]. Diverse classes of antihypertensive prescription may be used for blood pressure manage in diabetes among these angiotensin-II type 1 receptor blockers (ARBs), calcium channel blockers, thiazide diuretics and ACE inhibitors are common [120]. Cheung demonstrated that the calcium antagonists have been extensively used in hypertensive patients with diabetes [121]. Use of Verapamil a calcium channel blocker significantly reduced the risk of developing diabetes [122]. Similarly diabetic patients often take anti-hypertensive medications and coadministered with antidiabetic drugs [123]. Treatment of patients with hypertension and diabetes with ARBs improved both macrovascular and microvascular alterations [124].

Diverse classes of antihypertensive prescription may be used for blood pressure manage in diabetes among these calcium channel blockers, angiotensin-II type 1 receptor blockers (ARBs), thiazide diuretics and ACE inhibitors are common. Cheung demonstrated that calcium antagonists have been extensively used in hypertensive patients with diabetes. Collective pharmacological treatment generally entails in management of type 2 diabetes mellitus to attain satisfactory glucose manage and dealing of concomitant pathologies, drug–drug interactions must be cautiously considered with antihyperglycaemic drugs [125]*.* Mitra [126] conducted a study to examine the interaction of diabecon (D-400), a herbomineral anti-diabetic the most important purpose of this cram was to assess the "*in vitro"* drug interaction of enalapril, captopril and lisinopril with commonly prescribed antidiabetic drugs (metformin, pioglitazone glimepride and glibenclamide) by utilizing HPLC.

#### *3.4.1 Interaction of enalapril with antidiabetic drugs by HPLC*

*In vitro* interactions of enalapril in the presence of antidiabetic drugs (metformin, glibenclamide and glimepride) were carried out in 1:1 at 37°C and method for simultaneous determination of both interacting drugs was also developed as described in former sections. Results of these interactions are summarized in **Table 18** and plotted in **Figure 4**. The % availability of enalapril and metformin was found to be between 98 and 106% indicating no reaction between drugs. These results clearly indicated that enalapril could be safely co administered with metformin. The two drugs did not inhibit or disturb the absorption of each other. Similar behavior was observed with glibenclamide and glimepride, the availability of enalapril was found to be between 102 and 103% with glibenclamide and


#### **Table 18.**

*% availability of enalapril and antidiabetic drugs by HPLC.*

#### **Figure 4.** *% Availability of a inhibitors and antidiabetic drugs by HPLC.*

glimepride and the availability of glibenclamide and glimepride remained almost unchanged. No remarkable change in area under curve and drift in retention time were observed. However, the results showed that no interaction occurred as there % recovery remained almost unchanged.

#### *3.4.2 Interaction of captopril with antidiabetic drugs by HPLC*

In this study drugs were analyzed by measuring the area under curve (AUC), % recovery and considerable drift in retention time. Captopril and metformin did not affect the availabilities of each other i.e. 101% and 103% was observed respectively up to 30 minutes and at the end of experiment both were available up to 100% and 105% respectively. Similar effect was observed in presence of pioglitazone i.e. 102% of captopril, while 104% of pioglitazone was available at the end. In presence of glibenclamide, the %availability of captopril and glibenclamide were 102 and 101% at 30 minutes, which gradually increased and after 180 min were found to be103 and 106% respectively. Interacting results shows that no remarkable drifts in the availabilities and no drift in retention time were observed (**Table 19**). However the results showed that no interaction occurred as there was no significant change in % availabilities of both drugs were observed by HPLC.


*Interaction Studies of ACE Inhibitors with Antidiabetic Drugs DOI: http://dx.doi.org/10.5772/intechopen.99795*

#### **Table 19.**

*% availability of captopril and antidiabetic drugs by HPLC.*


**Table 20.**

*% Availability of lisinopril and antidiabetic drugs by HPLC.*

#### *3.4.3 Interaction of lisinopril with antidiabetic drugs by HPLC*

In this study drugs were analyzed by measuring the area under curve (AUC), % recovery and considerable drift in retention time. Presence of metformin, pioglitazone and glibenclamide could also not assert any significant change in availability of lisinopril at 37°C. Availability of lisinopril with metformin was 103.33 at the end of experiment and that of metformin was 104.33%. In presence of pioglitazone and glibenclamide 100.3 and 102% of drug was available at the end of experiment and the availability of pioglitazone and glibenclamide were also not affected in presence of lisinopril. The obtained results showed that the NIDDMs and lisinopril do not affect *in-vitro* availability of each other at 37°C (**Table 20**).

#### **4. Conclusions**

The method described is simple, universal, convenient and reproducible simultaneous method that can be used to determine and quantify ACE inhibitors and antidiabetic drugs. Reliability, rapidness, simplicity, sensitivity, economical nature, good recovery and precision of this method give it an advantage over the other reported HPLC methods for the determination of ACE inhibitors and antidiabetic drugs. In summary, the proposed method can be used for drug analysis in routine quality control. In addition, this method has wide application in clinical research and pharmacokinetics drug interactions.

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Safila Naveed\* and Halima Sadia Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jinnah University for Women, Karachi, Pakistan

\*Address all correspondence to: safila117@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 6**

## Combined Effect of Metformin and Statin

*Sabu Mandumpal Chacko and Priya Thambi Thekkekara*

#### **Abstract**

Diabetes mellitus (DM) is considered a risk factor for the development of coronary artery disease (CAD). Metformin, an anti-diabetic drug, has been shown to lower the cardiovascular events in pre-clinical and clinical studies. Many research articles suggests that metformin has a protective effect on CAD beyond its hypoglycemic effects. Patients with diabetes type 2 have an increased risk for cardiovascular disease and commonly use combination therapy consisting of the anti-diabetic drug metformin and a cholesterol-lowering statin. Statins have been found to be a safe and effective approach to reduce serum low density lipoprotein cholesterol (LDL-C) levels, which is the cornerstone for primary and secondary prevention of atherosclerosis. However, regular statin monotherapy in some patients may not be sufficient to achieve a therapeutic LDL-C. It has been reported that statins increased the incidence of new-onset diabetes in a dose dependent manner especially in women, the elderly, or in the presence of a family history of type 2 diabetes (T2D) and Asian ethnicity. The molecular mechanisms contributed to antioxidation, anti-inflammation, and anti-apoptosis. In this chapter, we aimed to investigate whether the combined administration of metformin and atorvastatin could achieve superior protective effects on different disease treatment purpose and to elucidate its molecular mechanisms of the combinations.

**Keywords:** combination therapy, metformin, statins, diabetes mellitus, clinical studies

#### **1. Introduction**

World Health Organization (WHO) defines diabetes mellitus as a metabolic disorder of multiple etiologies characterized by chronic hyperglycemia with alterations of carbohydrate absorption, fat and protein metabolism. DM is one of the four major non-communicable diseases along with cardiovascular disease (CVD), cancer and chronic respiratory diseases. Once a disease of affluence, it is now increasingly common among the poor countries [1]. The morbidity and mortality associated with DM arises from minor and macrovascular complications, ischemic heart disease (IHD) and peripheral vascular disease (PVD) [2]. Metformin acts by several mechanisms of action but the major mechanism is inhibiting hepatic gluconeogenesis [3]. The drug may antagonize the action of glucagon, and reduces fasting blood glucose (FBG) [4]. In addition, metformin increases insulin action at target sites, increases peripheral glucose uptake, enhances fatty acid oxidation and reduces glucose absorption from gastrointestinal tract [5]. Diabetes mellitus and statins have a complex association and are the attention of patient and healthcare

debate. Statins are widely used as a part of diabetes mellitus care due to that patients with DM have a greater CVD [6]. At the early stage, the heart only showed transcriptional and metabolic altercations, including enhanced inflammation, oxidative stress, depletion of antioxidant proteins, and changes in energy metabolism. Use of statins in diabetes is a controversial when compared with metformin. Although the potential detrimental effects of statin on muscle and liver have been known for a long time, new concerns have emerged regarding the risk of new onset diabetes (NOM). This often leads to discontinuation of statin, non-adherence to therapy, or concerns correlating with initiating statin therapy.

There are several CVD risk factors, including hypertension, dyslipidemia, diabetes mellitus (DM), smoking and obesity, as well as platelet dysfunction. Certain drugs are currently available for treating these risk factors, whereas drug combinations are frequently needed to achieve therapeutic goals especially in hypertension, DM and coronary heart disease (CHD). Based on these considerations our objectives were 1) to assess whether combination therapy shows clinical effectiveness for cognition and functional benefits in a well-characterized prospective cohort of patients with T2DM treated over years with metformin; 2) to determine the magnitude and duration of benefit; 3) to characterize the long-term treatment of patients who receive combination therapy compared to those who were never treated with statins and those who only received metformin as monotherapy; and 4) to use modeling methods to make predictions about the mechanism and clinical course in different treatment groups and dose levels.

Both metformin and statins thus act on glucose—as well as lipid metabolism which is why metformin–statin combination therapy is prescribed to many T2DM patients. Since both drugs act on glucose as well as lipid metabolism, it is important to understand in detail the interactions between metformin and statin mechanism of action on treatment design with different dose level and optimal safety/efficacy profiles. This chapter is therefore designed to provide insight in the mechanism of combined effect of statin/metformin not only on DM and CVD but also with different types of cancer and other diseases. This chapter also explain the interaction of both drugs on preclinical and clinical studies to determine an optimal dosing strategy of both drugs.

#### **2. Metformin**

Metformin is an oral antidiabetic drug, discovered in 1922, came on the market as late as 1979 [7]. The drug is belongs to the biguanide classification and derivative from guanidine found in *Galega officinalis*. It is available in different formulations based on its duration of action like immediate-release, extended release and delayed-release metformin [8, 9]. The latter two forms were developed to expand the absorption of metformin along the gut. Metformin administration in 30 min before a meal produced highest therapeutic efficacy in lowering postprandial hyperglycemia [10].

#### **2.1 Metformin absorption and distribution**

Oral administration of metformin transported into the small intestine across the apical membrane into the enterocytes via several transporter proteins. The main proteins are the plasma monoamine transporter (PMAT; SLC29A4), organic cation transporter 1 (OCT1; SLC22A1) and serotonin transporter protein (SERT; SLC6A4) [11].

Metformin accumulated majorly in the intestine, and in the stomach, liver, kidney and lesser extent in muscle. The accumulation of metformin in intestine

#### *Combined Effect of Metformin and Statin DOI: http://dx.doi.org/10.5772/intechopen.100894*

and stomach is because of these organs are most exposed to high concentrations of metformin after oral administration. A recent study confirmed the high metformin levels are accumulated in these organs [12]. These concentrations are tenfold higher than metformin concentrations in the liver, indicating that the intestine is probably an important site of action. In fact, the metformin effects in the intestine may be rather different than the effects in the liver. The concentration of metformin in human jejunum has been shown to be 30 to 300 fold greater than in plasma, and earlier studies demonstrating accumulation of metformin in the intestinal mucosa. Metformin navigates to the liver via the portal vein and is taken up predominantly by organic cation transporter (OCT1) as well as by Thiamine transporter (THTR-2). In this chapter, the effects of metformin on the lipid metabolism are highlighted, thereby creating a special focus on the effects on lipids related to the activation of AMPK by metformin (**Figure 1**) [13].

Metformin is transported into hepatocytes mainly via OCT1, and inhibited the mitochondrial respiratory chain (complex I) through a currently unknown mechanism(s). The deficit in energy production is balanced by reducing gluconeogenesis in the liver. This is mediated in two main ways. First, a decrease in ATP and a concomitantly increase in AMP concentration. Second, increased AMP levels function as a key signaling mediator to (1) allosterically inhibit cAMP–PKA signaling by suppression of adenylatecyclase, (2) allosterically inhibit FBPase, (3) activates AMPK. This leads to inhibition of gluconeogenesis (1 and 2) and lipid/cholesterol synthesis (3).

Metformin is present for over 99% in the mono protonated form in all tissues of the body except in the stomach. The sparse data showed, that metformin is mostly distributed in the cytosolic fraction (~ 70%) of rat hepatic cells compared to mixed membranes (12%), nucleus (~ 5%), and mitochondrial and lysosomal fractions (8%). A low binding affinity of metformin to mitochondrial membranes was seen, and this may be because of the two methyl groups present in metformin structure [14]. Previous study concludes that, the mitochondrial membrane

potential may promote entry of metformin (positively charged) [15], which will then concentrate inside the mitochondria (negatively charged) [16]. Molecular modeling of the metformin distribution and validation study confirmed the presence of high concentrations of the drug in the endoplasmic reticulum (ER) and in the mitochondria, based on its membrane potential [17].

#### **2.2 Metformin mechanisms on glucose and lipid metabolism**

The main mechanisms of metformin involved in decreasing the endogenous glucose production and plasma glucose have all been extensively reviewed and critically discussed in earlier studies [18]. Metformin shows beneficial effects on the glucose and lipid metabolism, even though the pathways are not fully understood [19]. In patient studies, the variations of metformin efficacy may be due to the presence of responders and non-responders to the drug treatment [20], racial and ethnic background [21], and personal variation in the adaptation of metformin treatment. Sonne et al., [22] proposed a pathway inducing reduction of LDL cholesterol by the. Inhibition of the intestinal absorption of bile acids is caused by metformin. It causes an increased synthesis of bile acids in the liver, and cholesterol is used for this process [23], thereby causing a decreased amount of cholesterol in the hepatic cells. Upregulation of the LDL-C receptor may increase the uptake of lipoproteins, to restore a sufficient level of cholesterol in the liver. Hence, metformin indirectly decrease the LDL-C concentration and plasma total cholesterol concentrations.

#### **2.3 (In)-direct effects of metformin on** β **cells**

A decreased β cell mass is an important factor in the development of T2DM. High glucose and FFA induce damaging effects on β cells (e.g. decreased insulin secretion and β cell mass) [24]. It is therefore of interest to consider possible beneficial effects of metformin on β cell function. Lipase and amylase are secreted by the pancreas and are often measured to monitor the condition of the pancreas. There were no changes observed in the enzyme levels, and the pancreas volume when metformin (1950 mg/day) was given to T2DM patients for 24 weeks. This works suggesting that metformin does not repair damaged β cells [25].

#### **3. Statin**

Statins, block an enzyme called HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase) that is involved in the synthesis of mevalonate, a naturally occurring substance that is then used by the body to make cholesterol. By inhibiting this enzyme, LDL-cholesterol and cholesterol production is decreased. Statins also increase the number of LDL receptors on liver cells, which increases the uptake and breakdown of LDL-cholesterol. Most of the effects of statins, including the blocking of the HMG-CoA reductase enzyme occur in the liver. Many research have shown that elevated levels of total cholesterol, LDL-cholesterol, triglycerides, and apolipoprotein B increase a person's risk of developing heart disease or having a stroke.

#### **3.1 Classification of statins and its general source**

Statins are classified based on different criteria, including: 1) how they are obtained, 2) liver metabolism, 3) physicochemical properties, and 4) specific activity. Some of the statins are obtained after fungal fermentation: lovastatin, *Combined Effect of Metformin and Statin DOI: http://dx.doi.org/10.5772/intechopen.100894*

pravastatin and simvastatin, others by synthesis: fluvastatin, atorvastatin, and cerivastatin. Only five statins are, at this moment, in clinical use: lovastatin, simvastatin, pravastatin, atorvastatin and fluvastatin. Pravastatin is extremely hydrophilic, fluvastatin has intermediate characteristics, lovastatin, simvastatin, atorvastatin and cerivastatin are hydrophobic.

#### **3.2 General uses of statins**


#### **3.3 Statins mechanism on glucose and lipid lowering metabolism**

Statins are a major class of drugs that decrease plasma cholesterol levels and are prescribed as first choice to patients suffering from CVD [26]. Simvastatin and atorvastatin are often given as a first choice to patients with cardiovascular risk factors/cardiovascular disease. In earlier studies reported that low dose (20 mg/day) of atorvastatin given to patients with myocardial infarction showed improved lipid, adipokine, and pro-inflammatory markers and decreased insulin resistance. Higher dose (40 mg/day) of atorvastatin showed hyperglycemia, increased leptin levels and ghrelin deficiency [27, 28] in diabetic patient. It was also discovered that the reduction in LDL-C by statins is an important indicator of increased T2DM risk [29]. Genetic factors and/orange-related factors could as well lead to the development of T2DM during statin treatment.

Several mechanisms possibly involved in the effect of statins on glucose metabolism are summarized in **Figure 2**. Statin signaling pathway that stimulates endogenous glucose production (EGP) by activation of gluconeogenic genes in human liver cells. Statin activates the pregnaneX receptor (PXR) in the cytoplasm. Many functions are exerts by PXR, such as the stimulation of the expression of proteins involved in regulation of hepatic glucose and removal of xenobiotics, and lipid metabolism [17].

#### **3.4 Effects of statins in the β cell of pancreas**

Statin mechanism may contribute to a decreased insulin secretion in the β cell, possibly contributing to the progress of T2DM. The upregulation of LDL-C receptor seen upon inhibition of HMG-CoA reductase are one of the directly

#### **Figure 2.**

*Hypercholesterolemia enhance the entry of LDL particles into sub endothelial space at lesion-prone arterial sites. Monocyte chemotactic protein-1 (MCP-1) and oxidized-LDL act as chemoattractants to direct accumulation of monocytes and their migration to the subendothelial space, where monocytes undergo phenotypic transformation into macrophages. Oxygen free radicals concurrently modify LDL. Oxidatively modified LDL is taken up by nondownregulating macrophage receptors to form lipid-rich foam cells. The foam cells develop into fatty streaks that is the, precursor of atherosclerotic plaques. Statins exhibit pleiotropic effects on many components of atherosclerosis that accompany hypercholesterolemia, abnormal endothelial function and including platelet coagulation abnormalities, and determinants of plaque thrombogenicity such as plaque inflammation and proliferation.*

affected processes, which results in increased uptake of plasma LDL-C into the β cell [30]. The increased amount of cholesterol within the cell causes interference with translocation of glucokinase, to the mitochondria [31]. A decreased glucose transporter (GLUT2) expression level was observed in simvastatin treated mouse MIN6 cells which resulted in a reduction of ATP levels. This may be the mechanism of inhibition of the KATP channel closure, membrane depolarization and calcium channel opening all leading to reduced insulin secretion [32]. Inhibition of the ATP-dependent potassium channel, depolarization and the decreased influx of intracellular calcium, and calcium concentrations were observed and were related to a decreased insulin secretion. In an ex vivo study, intracellular calcium levels were not affected even though intact with single-islets were treated with simvastatin [33]. Statin treatment may cause inactivation of Ras and Rho molecules, hence the activation and membrane translocalization of GLUT-4 is inhibited. Experiments with atorvastatin treatment in mouse adipocytes confirmed that GLUT-4 located on the plasma membrane moved to the cytosol during treatment and this may result in an increased insulin resistance [34].

#### **3.5 Statins on cancer**

Since 1959, evidence from many studies had revealed that there was an association between T2DM and cancer, and patients who had T2DM were more likely to be diagnosed with cancer than patients who had not [35, 36]. A lot of evidence has also shown its beneficial effects in cancers, including prostate, breast, lung, and colorectal cancers [37]. Experimental results in vitro have suggested the effect of statins on

*Combined Effect of Metformin and Statin DOI: http://dx.doi.org/10.5772/intechopen.100894*

growth, migration, apoptosis, and autophagy of cancer cells [38, 39]. The data from in vivo cell culture studies, statins may act as a preventive drug for hepatocellular carcinoma, malignant glioma and bladder cancer [40]. However, the role of statins on the incidence of cancer in patients with T2DM has not been well documented. Fei et al., [41] performed a meta-analysis to evaluate the impact of different types of statins on the risk of cancers with T2DM.The study was systematically searched with the Cochrane Library, PubMed, Embase, and Wanfang databases from January 1999 to March 2017. A pairwise meta-analysis used to estimate the pooled ratios (ORs) and 95% confidence intervals (CIs). NMA was performed to compare different types of statins. In pairwise meta-analysis result showed that, the incidence of cancer in T2DM patients was reduced when simvastatin, atorvastatin, pravastatin, fluvastatin, lovastatin, rosuvastatin, and pitavastatin were used. The analyses suggest that rosuvastatin may be more effective than others.

#### **4. Combination therapy of metformin and atorvastatin**

#### **4.1 On antidiabetic activity-preclinical studies**

Previous studies on diabetic rats (200–220 g) reported that after 2 weeks of metformin–atorvastatin combination therapy (500 mg metformin and 20 mg atorvastatin per 70 kg body weight), reduced blood glucose, lipid-lowering effects, and reduced in elevated oxidative stress, and positive effects on cardiovascular hypertrophy occurred [42]. The reduction of oxidative stress and liver protection (blood analysis and liver histology studies, e.g. CRP, TNF-α, IL-6, protein carbonyl levels) was also seen in T2DM rats treated with metformin and atorvastatin [43].

Statins consistently showed a protective role in the setting of diabetes cardiomyopathy (DCM) due to their roles of anti-inflammation, anti-oxidation, and antiapoptosis effects [44]. In previous animal experiments, statins could prevent DCM by all evicting left ventricular dysfunction and inhibiting myocardial fibrosis through anti-apoptosis and anti-inflammation pathways. It seems that statins may facilitate the onset of diabetes by impacting peripheral insulin sensitivity and islet b-cell function, while statins can effectively modify the promotive factors and promoting DCM, including inflammation and oxidative stress, thereby protecting the heart against diabetic conditions [45].

#### **4.2 On Antiatherosclerogenic activity-preclinical studies**

An animal study was designed to evaluate the effectiveness safety and mechanism of an atorvastatin/metformin combination therapy in a rabbit atherosclerosis model induced by a high-cholesterol diet. At the end of the experiment, all rabbits were sacrificed by injection of an overdose of sodium pentobarbital solution and the aortas were separated from the surrounding tissues. From the initiation of the aortic arch, 0.5 cm sections were excised for paraffin treatment [46] and the remaining aortas were soaked in 4%paraformaldehyde and then stained with Oil Red O solution, to evaluate the atherosclerotic lesion area of the aorta by image-processing software (ImageJ). One portion stained with hematoxylin and eosin (H&E) before quantification using ImageJ software. In an animal study 12-week high-cholesterol diet induced a significant increase in atherosclerotic lesion area in rabbits in the control (Ctrl) group; after 10 weeks of atorvastatin or metformin treatment, the atherosclerotic lesion area was significantly reduced by 51% and 35%, respectively.

Atorvastatin/metformin combination therapy resulted in an 80% reduction of atherosclerotic plaques compared with the control group. The combination

therapy showed which was more effectively than each monotherapy. Compared with control group, the treatment of atorvastatin or metformin significantly reduced the lesion size by 68% and 42%, respectively, while atorvastatin/ metformin combination therapy further reduced atherosclerotic lesion size by 86%. It was reported that large HDL is inversely associated with cardiovascular disease [47]. The results suggest that atorvastatin and metformin combination therapy is superior to atorvastatin monotherapy for the treatment of atherosclerosis and the underlying mechanisms might be associated with cholesterol efflux in macrophages. The study results demonstrated that atorvastatin/ metformin combination therapy did not show a better lipid-lowering effect than atorvastatin, which is similar with the recent clinical and preclinical data [48]. The CAMERA study revealed that metformin did not affect the lipid profile in statin-treated patients [49]. Forouzandeh *et al*. confirmed the plasma cholesterol in apoE−/− mice fed a high-fat diet did not affect and found that metformin markedly reduced atherosclerotic plaques [50]. Earlier studies also suggest that an additional anti-atherosclerotic mechanism of metformin when added to atorvastatin, which is independent of the lipid-lowering effect. Study report is the first, to demonstrate that atorvastatin/metformin combination therapy increases the percentage of large HDL sub fraction. Goldberg *et al*. [51] found that metformin could raise the concentrations of large HDL in a clinical trial. The research article also suggested an inverse association of large HDL sub fraction with coronary artery disease, which may involve reverse cholesterol transport (RCT).

#### **4.3 On antidiabetic activity-clinical studies**

In a clinical study a great number of patients are selected and treated with metformin–atorvastatin combination tablet administered as a single daily dose [52]. There is only a minor chance for toxic drug interactions when treated with metformin and statin together because metformin is not metabolized and is the mechanism for most statins are via the cytochrome P450 system [53]. Since metformin shows beneficial effects on both dyslipidemia and glycemic control and has been shown to reduce CVD risk while statins may have an added beneficial effect on CVD risk. Hence the combined treatment with both drugs seems a good option. Clinical studies on the effects of metformin and statin combination therapy have been carried out but for different diabetic complications [54–56]. Each of these studies had different objectives and included different patients groups, i.e. either with T2DM, dyslipidemia, treated (different doses), untreated, or newly diagnosed T2DM. This criteria were compared in these studies to arrive at overall results of metformin statin combination therapy. The lowest dose of metformin (500 mg) and atorvastatin (10 mg) once daily resulted in the highest reduction of fasting plasma glucose (−35%). Atorvastatin 20 mg showed to attenuate the glucose and HbA1c-lowering effect in combination with 1000 and 2000 mg metformin.

In another clinical trial, a total of 50 newly diagnosed patients with T2DM with age range of 47.8 ± 7.4 years and prescribed 850 mg/day of metformin (sustained release), with dietary restriction, were enrolled in open-label multi center pilot study. WHO criteria was followed for the selection of newly diagnosed patients [57] and underwent a physical examination and information about their medical history, demographic parameters, and medication history were obtained by questionnaire. The patients received a constant dose regimen of metformin during the 90-day study period. In that study, the use of metformin in newly diagnosed T2DM patients, improves body weight and glycemic control; however, the addition of

#### *Combined Effect of Metformin and Statin DOI: http://dx.doi.org/10.5772/intechopen.100894*

low-dose atorvastatin did not improve these conditions. Metformin, in a long-term study, reduces the risk of macrovascular disease after a follow-up period of 4 years [58], and this beneficial effect supports to continue metformin treatment with T2DM patients unless contraindicated. The result of this study is consistent with that reported in an experimental animal model, which indicates that the combination of atorvastatin with metformin did not produce a better lipid-lowering effect than atorvastatin [59]. In addition, the study indicated that 10 mg/day did not increase the HbA1c and serum glucose levels, but there was no additional significant improvement in the studied markers when compared with the metformintreated group.

#### **4.4 On lipid metabolism -clinical studies**

The effects of metformin on lipid homeostasis discussed earlier in this chapter, indicate that lipid metabolism is positively affected in the intestine and liver leading to decreased plasma triglycerides, LDL-C, and total cholesterol. Metformin effects on lipid metabolism seem to be localized to the intestine. Statins mainly act on plasma cholesterol via activation of the LDL-receptor suggesting that combination therapy should show an additional effect on plasma lipids. Combination therapy with statins and metformin demonstrated beneficial effects in patients with other disease(s)/disorder(s) than T2DM and dyslipidemia [60].

In earlier studies, the effect of metformin alone on the lipid profile was studied, and the result analysis showed that only TG levels and LDL/HDL ratio were significantly improved. Whereas these effects were not significantly different compared with its combination with atorvastatin that improves all lipid profile components. These results indicated that the addition of atorvastatin with metformin did not influence the lipid-lowering effects of monotherapy in newly diagnosed T2DM patients with metformin. In previous studies, although metformin moderately improves the lipid profile, there were inconsistencies in its effects on the lipid parameters [61]. Accordingly, the addition of atorvastatin to metformin treatment in newly diagnosed T2DM patients showed relatively normal lipid profile may be irrational and cost ineffective and the emergence of adverse effects may be highly expected with long-term use.

#### **4.5 On prostrate cancer-clinical studies**

Diabetic patients receiving metformin have been shown to have a reduced cancer incidence and a decrease in cancer-specific mortality [62]. Statin use was also found to be associated with a reduction in the risk of biochemical recurrence in patients with prostate cancer and a decreased risk of cancer mortality [63, 64]. Based on epidemiologic evidence and the preclinical data for metformin and atorvastatin individually in prostate cancer, the author concluded the beneficial effects of metformin and atorvastatin alone or in combination on SCID mice and cultured prostate cancer cells. Metformin and atorvastatin in combination exhibited potent inhibitory effect on the growth of prostate cancer cells *in vivo* and *in vitro*. The drug combination stimulated apoptosis in prostate cancer cells compared with individual treatment. Mariel concluded that, coupled with epidemiological studies, provide a strong rationale for clinically evaluating the combination of metformin and atorvastatin in prostate cancer patients [17].Recent studies showed that metformin in combination with simvastatin induced G1-phase cell cycle arrest, and Ripk1- and Ripk3-dependent necrosis in prostate cancer cells [65, 66]. The combination of metformin and simvastatin was found to decrease the levels of phospho-Akt and phospho-AMPK*α*1/*α*2 [67].

#### **4.6 Combination therapy on other diseases**

In T2DM patients with non-alcoholic fatty liver disease (NAFLD) the combination therapy was found to be benefited. Whereas, statin therapy associates negatively with non-alcoholic steatohepatitis and found to be significant fibrosis while a safe use of metformin in patients with T2DM and NAFLD was demonstrated [68]. Combination therapy consisting of metformin and statin treatment is frequently prescribed to women with polycystic ovary syndrome (PCOS). This syndrome increases the risk of T2DM and cardiovascular morbidity as it is associated with abnormal increased lipid levels, insulin resistance, endothelial dysfunction and systemic inflammation [69]. Meta-analysis showed that combined therapy in women with PCOS resulted in improved inflammation and lipid markers but it did not improve insulin sensitivity [70].

Treatments using statins, and combined statins and metformin can effectively improve IR, fasting insulin (F-INS), insulin sensitivity index, hyperandrogenemia, acne, hirsutism, testosterone and decreasing C reactive protein (CRP) [71–73]. Pre-treatment with atorvastatin for 3 months followed by metformin in patients with PCOS improves insulin and homeostasis model assessment of IR (HOMA-IR) indices and reduces CRP level but does not improve the lipid profile compared with placebo treatment. Hence, atorvastatin pre-treatment enhances the effects of metformin in improving IR, whereas inflammatory markers are not affected by decreased total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) after cessation of atorvastatin [74].

The lipid-lowering effect of statins administered with or without metformin in PCOS patients remains ambiguous. This finding is also supported with the metaanalysis performed by Gao et al. [75]. A clinical trial demonstrated that insulin secretion was found to be increased after 6 weeks of statin therapy in women with PCOS [76]. The meta-analysis found that statins fail to improve F-INS and HOMA-IR in single or in combination with metformin. This finding may be due to the following reasons. First, statins may damage endothelial function through loss of the protective anti-proliferative and anti-angiogenic effects of adiponectin, resulting in impaired insulin sensitivity [77]. Second, statins decrease the levels of cholesterol mediated by the farnesoid X receptor (FXR), the deficiency of which is related to IR [78]. The activation of FXR can lower the levels of glucose-6-phosphatase, reduce phosphoenol pyruvate carboxykinase in gluconeogenesis, and increase glycogen synthesis [79]. Hence, induced IR caused by statin therapy may be related to the low expression of FXR [80]. Third, statins (lipophilic) are possibly absorbed by extra-hepatic cells; these statins can deregulate cholesterol metabolism, thus deteriorating IR and attenuating β-cell function [81].

Combination therapy could also be considered for T2DM patients with diabetic retinopathy. Diabetic retinopathy (DR) is a microvascular complication of diabetes caused by hyperglycemia and hyperosmolarity. In T2DM patients and pre-existing DR patients, the use of statin showed a protective effect against development of diabetic macular edema [82]. In T2DM patients receiving statin therapy in combination with increased levels of cholesterol remnants and triglycerides were associated with slight decreased in left ventricular systolic function. Targeting cholesterol remnants might be beneficial for finding cardiac function in T2DM patients receiving statins [83].

#### **5. Combination therapy of metformin and simvastatin- clinical studies**

A high daily dose of metformin (3000 mg) and simvastatin (40 mg) resulted in an improved insulin resistance, but fasting plasma glucose decreased only by 5%,

#### *Combined Effect of Metformin and Statin DOI: http://dx.doi.org/10.5772/intechopen.100894*

and observed minor changes on lipid metabolism parameters. This may probably due to the fact that metformin was given on top of simvastatin treatment. The patients involved in these studies had an impaired fasting glucose, dyslipidemia, newly diagnosed T2DM and/or dyslipidemia. However, it could be used for hypothesis-generation rather than making rigid decisions, considering the lack of multiple dose dependent combination studies.

The combination of metformin with insulin may be a better therapeutic option for patients with DM whose hyperglycemia is poorly controlled on insulin treatment. Aviles et al. [84] stated that increased frequency of dosage of insulin causes more improvement in glycemic control and significantly reduce HbA1c which was compared with a combination therapy of insulin and metformin. Furthermore, unchanged FBG and PPBG and HbA1c in patients on metformin and insulin compared to combination of metformin, insulin and simvastatin treated patients. The HbA1c of diabetic patients on simvastatin showed a slight elevation as compared to other groups. Previous studies reported that statin use is associated with a rise of FPG in patients with and without DM [85]. Sattar et al., have identified deterioration in glucose homoeostasis in patients treated with statins and this depends on lipid solubility of statins. Simvastatin can enter easily extra hepatic cells because of its high lipid solubility and may suppress isoprenoid protein synthesis, thus attenuating the action of insulin. The abnormal level of FBG may translate into clinical syndrome of DM with rise in HbA1c is not excluded. The combination of metformin and insulin may be an attractive therapeutic option for patients with DM whose hyperglycemia is poorly controlled on insulin [86].

#### **6. Conclusion**

The mechanism of metformin is a controversial along with the use of statins in diabetes. Although the potential detrimental effects of statin therapy on muscle and liver have been known for a long time, new concerns have emerged regarding the risk of new onset diabetes (NOM) that often leads to discontinuation of statin, concerns correlating with initiating statin therapy or non-adherence to therapy.

Metformin is generally to exert its beneficial effects on glucose metabolism mainly in the liver. In line with recent research articles on the topic we conclude that the drug acts primarily in the intestine. This is due to the at least one order of magnitude higher concentrations of metformin in the intestine than in the liver. The drug present in the liver and its effects may be localized to this organ most probably via its effects on gluconeogenesis. A newly diagnosed patient with T2DM who show inadequate response to metformin may need better treatment approaches to lower atherogenic lipids. Supplementation with niacin or high-dose omega-3 fatty acid could be used in newly diagnosed T2DM patients with borderline values of lipid profile, secondary to lifestyle modifications before using a potent statin such as atorvastatin as the first treatment priority.

The effects of metformin on lipid metabolism as discussed in this chapter indicate that lipid level is positively affected in the intestine and liver leading to decreased LDL-C, plasma triglycerides and total cholesterol. Metformin effects on lipid metabolism seem to be localized to the intestine. Statins mainly act on plasma cholesterol levels via activation of the LDL-receptor suggesting that combination therapy should show an additional effect on plasma lipids. This may influence glucose homeostasis primarily by inhibition of insulin secretion in pancreatic β cells. T2DM patients receiving statin therapy in combination, with increased levels of cholesterol remnants and triglycerides were associated with slight decreased in left ventricular systolic function. Targeting cholesterol remnants in addition

to T2DM patients receiving statins might be shown beneficial effect on patient's cardiac function. To treat T2DM and its secondary complications, the combination therapy of metformin with statins seems well placed and may act as a double-sided sword particularly in the case of statins. Whereas, statins alone increases the risk on T2DM particularly in pre-diabetic subjects, and co-treatment with metformin might reduce this risk.

We have concluded that, previous studies investigated possible sites of interaction of metformin and statins and they act on largely parallel pathways. Many studies suggested that the benefits of statin therapy for diabetes far outweigh any real or perceived risks, not suggested/recommended for discontinuation of statins for diabetic patients. In conclusion, both metformin and atorvastatin can protect DCM via the mechanism of anti-inflammation and anti-apoptosis activities. The combined administration of metformin and atorvastatin resulted in superior protective effects on DCM than a single drug treatment. In this chapter, we have compiled the possible sites of interaction of metformin and statins and conclude that they act on largely parallel pathways.

#### **Conflict of interest**

The authors declare no conflict of interest among themselves.

#### **Abbreviations**


*Combined Effect of Metformin and Statin DOI: http://dx.doi.org/10.5772/intechopen.100894*


### **Author details**

Sabu Mandumpal Chacko1 \* and Priya Thambi Thekkekara2

1 Mookambika College of Pharmaceutical Sciences and Research, Muvattupuzha, Kerala, India

2 Department of Chemistry, Baselius College, Kottayam, Kerala, India

\*Address all correspondence to: mcsabu74@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 4
