Polypharmacology of clinical sodium glucose co‐transport protein 2 inhibitors and relationship to suspected adverse drug reactions

Abstract Sodium glucose co‐transporter 2 inhibitors (SGLT2i) are a promising second‐line treatment strategy for type 2 diabetes mellitus (T2DM) with a developing landscape of both beneficial cardio‐ and nephroprotective properties and emerging adverse drug reactions (ADRs) including diabetic ketoacidosis (DKA), genetic mycotic infections, and amputations among others. A national register study (MHRA Yellow Card, UK) was used to quantify the SGLT2i's suspected ADRs relative to their Rx rate (OpenPrescribing, UK). The polypharmacology profiles of SGLT2i were data‐mined (ChEMBL) for the first time. The ADR reports (n = 3629) and prescribing numbers (Rx n = 5,813,325) for each SGLT2i in the United Kingdom (from launch date to the beginning December 2019) were determined. Empagliflozin possesses the most selective SGLT2/SGLT1 inhibition profile at ~2500‐fold, ~10‐fold more selective than cangliflozin (~260‐fold). Canagliflozin was found to also inhibit CYP at clinically achievable concentrations. We find that for overall ADR rates, empagliflozin versus dapagliflozin and empagliflozin versus canagliflozin are statistically significant (χ 2, p < .05), while dapagliflozin versus canagliflozin is not. In terms of overall ADRs, there is a greater relative rate for canagliflozin > dapagliflozin > empagliflozin. For fatalities, there is a greater relative rate for dapagliflozin > canagliflozin > empagliflozin. An organ classification that resulted in a statistically significant difference between SGLT2i was suspected infection/infestation ADRs between empagliflozin and dapagliflozin. Our findings at this stage of SGLT2i usage in the United Kingdom suggest that empagliflozin, the most selective SGLT2i, had the lowest suspected ADR incident rate (relative to prescribing) and in all reported classes of ADRs identified including infections, amputations, and DKA.


| INTRODUC TI ON
Sodium glucose co-transporter 2 inhibitors (SGLT2i) are a relatively new class of antidiabetic medication for type 2 diabetes. 1 In comparison to other antidiabetic medication, 3 the pharmacological action of SGLT2i works independently of insulin. [4][5][6] SGLT2i competitively bind to SGLT2, are predominantly found in the kidneys, and are responsible for 90% of glucose reabsorption in the body, leading to the inhibition of this transporter. This consequently results in glucosuria and beneficial changes can be observed metabolically and hemodynamically in the body. 5,7,8 The inhibition of SGLT2 leads to the reduction in blood pressure, body weight, 9 and increase in ketone production. The increase in plasma ketones is associated with increased glucagon secretion from the pancreas, as a response to reduced glucose and lipolysis.
The heart utilizes the ketones for fuel and produces more ATP per molecule compared to glucose, decreasing oxidative stress. Diabetes is associated with both macrovascular and microvascular complications, an example being diabetic nephropathy which is caused by damage to blood vessels due to high blood glucose. Another indirect action of SGLT2 inhibition is natriuresis; this further reduces blood pressure and slows down the progression of chronic kidney disease (CKD). 6 Thus, SGLT2i have both cardioprotective and nephroprotective features. 7,8,10 This has been supported by the following EMPA-REG OUTCOME (Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients), DAPA-HF (Dapaglifozin and Prevention of Adverse Outcomes in Heart Failure), and CANVAS (the CANagliflozin cardioVascular Assessment Study) trials. 5,6,[11][12][13][14][15] Collectively, the results of the landmark trials demonstrated that SGLT2i correspond to a reduction in all cardiovascular mortality risks, reduced heart failure-associated hospitalization, and decline in CKD progression in diabetic patients. 16 These properties were identified to be shared across the SGLT2i class and therefore highlight the potential use in other co-morbidities including non-diabetic CKD. 17 Adverse drug reactions (ADRs) are defined as harmful and unintended events in response to a drug related to any dose. 18 SGLT2i are associated with ADRs 19 such as diabetic ketoacidosis (DKA), 20 acute kidney injury (AKI), genital mycotic infections, limb amputations and emerging links to stroke, 21 and bladder cancer. 22,23 However, there is no research regarding the polypharmacology of SGLT2i 24,25 and their suspected ADRs in pharmacovigilance studies. 26,27 This research aims to identify potential links between ADRs for the three most established SGLT2i in the United Kingdom with their unique polypharmacology and physicochemical profiles.
Our continuing research interest in the intersection of medicinal chemistry 28 with clinical prescribing and associated ADRs [29][30][31] warranted an application of our techniques for understanding the potential links between the SGLT2i's polypharmacology and ADRs landscape. Furthermore, recent studies have revealed that drugs believed to be highly selective frequently address multiple target proteins 32 and this may affect SGLT2i performance 33 and could lead to a more stratified approach in clinical practice.

| Chemical and pharmacokinetic properties of SGLT2 inhibitors
Physicochemical properties are intrinsic to each SGLT2i and have an impact on how they behave in the body including permeability, clearance, and absorption.
The Electronic Medicines Compendium 34 and ChEMBL database 35-38 were used to identify the chemical properties and pharmacokinetics of the SGLT2i (Table 1). Physicochemical properties including molecular weight, pKa, Log 10 P, and Log 10 D values were datamined from ChEMBL using the search terms of each drug's name. Calculated properties were obtained using the ChemDraw 19.0 software based on the chemical structure of each drug.
Neutral or basic-acidic drug molecules are defined by the pK a (negative base10 logarithm of the acid dissociation constant; the lower the pK a value the stronger the acid); log 10 P (where P is the partition coefficient = concentration of solute in octanol divided by the concentration of solute in water); log 10 D is the distribution constant of a drug between the aqueous and lipid phases at pH 7.4.
The following parameters were calculated; pIC 50 was calculated using the median SGLT2 inhibitory IC 50 of each drug; and lipophilic ligand efficiency (LLE) was calculated: LLE = pIC 50 -clog 10 P. Lipophilic ligand efficiency (LLE) measures how effective a drug binds to its target excluding nonspecific entropic factors. The LLE parameter is used to normalize potency relative to lipophilicity. A value of <5 is associated with increased toxicity. [39][40][41] The threshold for BBB penetration was set as: molecular weight <450 Da; <6 hydrogen bond donors (HBD); <2 hydrogen bond acceptors (HBA); neutral or basic drug molecule (defined by pKa); topological polar surface area ( t PSA) <90 Å; logD 7.4 1-3; and low affinity to efflux ABCB1 (P-glycoprotein, MDR1). The C max peak serum concentration of each SGLT2i was calculated from the FDA new drug application documentation. [42][43][44][45] Non-listed pharmacokinetic parameters on the ChEMBL database were collated through literature database searches of each drug name + pharmacokinetic OR PK search terms using Springer Link; NCBI; the electronic Medicines Compendium (eMC); and SciFinder (Caplus and Medline databases). 46-52

| Pharmacological properties of SGLT2 inhibitors
Pharmacological bioactivity data were curated. The ChEMBL database (accessed on 01/11/2020) was used to gather quantitative measures between each SGLT2i and human proteins. SGLT2 activity data were also identified through cross-checking literature using the Reaxys ® database. Bioactivity was compared using IC 50 values, with a minimum threshold set at 10 μM (to exclude weak interactions). IC 50 is a quantitative measure that indicates how much of a substance (in this case a drug) is required to inhibit a biological component by 50%.
The mean IC 50 gives an overview of the relative affinity between SGLT2i. The ADR event data collection was terminated in January 2020 due to differences in reviewal dates between the Yellow Card Report Scheme and Open Prescribing.
The reporting system processed reports for all SGLT2i except for ertugliflozin. Ertugliflozin has only recently been approved for use in clinical practice (2019), and thus, no ADR data was available at the time of data collection. The Yellow Card Report scheme categorized ADRs by organ class, which was replicated in this study.
Significant ADRs were selected and assessed within this study.
The selection criteria included differential ADRs across the SGLT2i (independent of ADR level above baseline) or high levels of ADR within a particular organ class (above baseline). The ADR table included ADRs that are established and suspected ADRs that were reported (n > 50) for each SGLT2i (Chart S1). ADRs reported under the investigations system organ class, non-serious ADRs, or ADRs because of multiple active constituents were removed to avoid biases.
The ADR data required standardization to allow for comparisons between the different drugs. ADRs per 100,000 R x is a standard approach in signal hypothesis generation. 29,31

| Prescribing data
The Open Prescribing (https://openp rescr ibing.net/) database collects data regarding prescription numbers for all prescribed drugs in England across all National Health Services (NHS) in primary care Clearance, a measure of how rapidly a drug is excreted; HB, hydrogen bond(s); t PSA, topological polar surface area of the compound.

TA B L E 1
The physiochemical and pharmacokinetic properties for the four SGLT2i settings. 56 The data for prescription numbers were extracted be-

| Statistical analysis
Chi-squared (χ 2 ) test was performed on the standardized ADR/100,000R x data to determine statistically significant differences between the suspected ADRs and SGLT2i using Excel for Microsoft 365. A p-value of <.05 was considered statistically significant. 57 Ertugliflozin was excluded from this part of the study due to the small number of reports. 58 Statistical analysis was performed on the three established SGLT2i (empagliflozin, dapagliflozin, and canagliflozin). As we are interested in (1) differences between the SGLT2i and not as to whether a particular ADR is related to SGLT2i therapy and (2) because of the exploratory nature of this study, the lack of data on potential confounders, and the relatively low incidence of some of the ADRs, we used raw data rather than disproportionate analysis. The exploratory nature of this study also meant that corrections for multiple comparisons were not used.

| Ethic approvals and consent to participate
This study was conducted using publicly available data and no patient identifiable data was used. Thus, there was no requirement for ethical approval and approval of consent was required.

| Chemical properties and pharmacokinetics
The physical properties of all SGLT2i are similar, all being C-glycosides ( Figure 1). They have prolonged action in the body due to the long t ½ and decreased metabolism in the gastrointestinal (GI) system.
All SGLT2i are given orally and as single-dose medication ( Table 1).
the exception being dapagliflozin which has V d of 118 L indicating its presence in tissues is higher than other class members.

| Pharmacological properties
IC 50 > 10 µM is considered a weak inhibitor and thus is clinically unachievable in most scenarios. SGLT2i C max range from 186 to 387 nM except for canagliflozin (2.8 µM).
Exhaustive data regarding the polypharmacology of the SGLT2i are available in Table S2 and key data are presented in Table 2. Selectivity values were approximations that compared inhibitory activity at SGLT2 versus SGLT1. Differences in selectivity between SGLT2is were noted. 59,60 Canagliflozin, selectivity being around 260-fold and can be considered the least selective and empagliflozin, being the most selective ~2500-fold for SGLT2 (Table 2).
Based on the physicochemical and polypharmacological data available, the following predictions can be made: empagliflozin is likely to emerge as the SGLT2i with the least number of serious ADRs due to high selectivity for SGLT2.  However, empagliflozin and dapagliflozin use was associated with foot, toe, and leg amputations at a relatively low incidence. The ADR incident range for toe and leg amputations for both empagliflozin and dapagliflozin was 0.24-0.25 and 0.035-0.14, respectively.

| ADR summary
For all ADRs relative to the nervous system, dapagliflozin (7.59) and canagliflozin (7.78) had a two-fold higher incidence of ADRs

| DISCUSS ION
The aim of this research was to identify potential relationships between the pharmacological activity of SGLT2i with their suspected ADRs. Visual inspection of Table 3

| SGLT2 inhibition in relation to ADRs
The polypharmacology of SGLT2i was predominantly of other SGLT transporters. In total, there are six SGLT transporters all coded by the SLC5A gene, but their functions and whereabouts in the body differ. 64 SGLT1 is located in the kidneys and is also present in the heart, small intestine, brain, and liver. In the kidneys, SGLT1 is responsible for 10% of glucose reabsorption from the urine. SGLT1 inhibition in the intestine is associated with GI side effects. 65,66 However, the roles of SGLT1 in other organs are not well understood.
The roles of the remaining SGLT transporters (SGLT4-6) are not fully elucidated to the best of our knowledge except for SGLT3 which is not a monosaccharide transporter but instead a sensor in the brain. 67,68 Therefore, no potential relationships can be drawn between the inhibition of SGLT1 at different organ levels (except the kidneys) and other SGLT transporters with their suspected adverse effects.
A recent study proposed that as a compensatory effect of direct SGLT2 inhibition, SGLT1 is upregulated in the kidneys and becomes responsible for approximately 40% of glucose reabsorption rather than the accepted 10%. 65,66 Due to the pharmacological activity of SGLT2i on SGLT1, SGLT1 inhibition may eventually lead to an increased glucosuria effect that could potentially be related to numerous complications such as ketoacidosis, limb amputations, and genital mycotic infections. The intensity of glucosuria is therefore related to the selectivity profile for SGLT2i. Canagliflozin's low selectivity due to its strong inhibitory effect on SGLT1 may correspond to an intense glucosuria effect. Whereas empagliflozin's high selectivity for SGLT2 may correspond to the lower comparative intensity in glucosuria, which is supported by the lower ADR incident rate for all ADRs, 44.07 and p < .05 (Table 3).
Complications that may arise due to increased urinary glucose excretion due to inhibition of both SGLT2 and SGLT1 eventually lead to indirect changes in the body. The indirect action of SGLT2 inhibition in the kidneys is associated with natriuresis. 4 As SGLT1's pharmacological action in the kidneys is analogous to SGLT2, inhibition of both could lead to potential increase in natriuresis. As a result of osmotic diuresis, loss of plasma volume and thus a reduction in blood pressure occurs. Therefore, this could explain the increased lower limb amputation rate associated with SGLT2i that is the result of hypovolemia and decreased tissue perfusion. Despite the association with all SGLT2i, canagliflozin had a greater relative amputation rate (compared to other SGLT2i) potentially due to its SGLT1 potent inhibition. [69][70][71] Furthermore, higher SGLT1 inhibitory activity with an SGLT2i corresponds to a higher infection rate which can be observed in Table 3 that displays canagliflozin possess the highest incident rate.
The potential mechanism that leads to a greater incidence in infection, both urinary tract and fungal infections, could be related to increase glucose presence in the urine. The environment is more nutrient rich and preferable for bacteria growth in the urinary tract and for fungus. Coupled with other urinary disorders such as urine dysfunction, polyuria, nocturia, incomplete bladder emptying, and weakened immune system due to diabetes may explain the high incidence of infection. 72 The concomitant usage of dipeptidyl peptidase-4 inhibitors is known to moderate the risk of genitourinary tract infections with SGLT2i use. 73 An increase in glucose excretion in urine is indirectly related to an increase in glucagon secretion from alpha cells in the pancreas.
This response is due to negative feedback in glucose control, reduced plasma glucose leads to glucagon secretion, and is an indirect action of SGLT2i. Glucagon is responsible for gluconeogenesis and glycolysis to regulate plasma glucose. The by-product of this is an increase in ketones in the plasma. Elevated ketones in the plasma elicit oxidative stress; other inflammatory responses thus can be potentially fatal. Other concerns include metabolic acidosis, as ketones remove bicarbonates in the body which is potentially hazardous. 74,75 The Yellow Card pharmacovigilance scheme has already provided an important safety update regarding SGLT2i and ketone levels. 76 SGLT2 inhibitor treatment should be interrupted in patients who are hospitalized for major surgical procedures or acute serious medical illnesses and ketone levels measured, preferably in blood rather than urine. Treatment may be restarted when the ketone values are normal and the patient's condition has stabilized.
Euglycemic ketoacidosis is a type of DKA and is a mixture of metabolic acidosis and hyperglycemia. 77 The relevance of increased ketone production in relation to SGLT2i despite it being prevalent for all is that dapagliflozin directly increases glucagon secretion in hyperglycemic conditions. This was observed in a study that compared glucagon secretion in mouse cells using canagliflozin and dapagliflozin. The pancreatic cells in mice are like humans. In both human and mice cells, SGLT1 is expressed on the alpha cells and inhibition of which suppresses glucagon secretion and was observed with canagliflozin due to potent inhibitory effect of SGLT1. 78,79 Dapagliflozin directly causing glucagon secretion from the pancreas may explain why the incident rate for DKA and total metabolic disorders were the highest, 16.09 and 25.54, respectively (Table 3). All SGLT2i indirectly cause glucagon secretion, due to urinary glucose excretion.

| Other transporter inhibition in relation to ADRs
All  (Table 1). Normally, the inhibition of CYP enzymes eventually leads to an increase in half-life, so the drug will stay present in the body longer and produces an extended pharmacological action.
Inhibition would also precipitate drug to drug interactions as CYP enzymes are responsible for the elimination of most drugs.
In addition, dapagliflozin inhibited organic anionic transporter polymorph (OATP1B3) which is responsible for numerous drugs transportation in the body and consequences of inhibition may lead to increase drug to drug interactions. 82 This poses as a possible risk in prescribing in clinical practice, as complications of drug-drug interactions can vary.
Furthermore, OATP1B3 is a transporter for bilirubin and inhibition of which is related to hyperbilirubinemia can lead to jaundice, cholestasis, and abnormal liver function. Hyperbilirubinemia is associated with neurological damage and can be fatal. This may explain why incidence of neurological disorders and hepatobiliary disorders are more prevalent with dapagliflozin than other SGLT2i (Table 3).
Furthermore, as glucose is the main metabolite for the brain for energy, varying plasma glucose may have negative implications on the nervous system. to the underestimation of any given reaction or drug even within the same class. 83 Furthermore, publicity about an adverse effect, 84 length of time on the market, and novelty of the drug may also affect reporting. This can mean that comparisons between drugs using such reports can be problematic, particularly when small numbers are involved. Declines in reporting ADRs after the second year of a drug on the market, known as the Weber effect, have been reported. 85 Reporters are requested to report any suspected ADRs, and they do not have to demonstrate a clear causal link with the drug.

| Limitations
These reported adverse reactions have not been proven to be related to the SGLT2i and should not be interpreted as a list of known side effects. Care needs to be taken with suspected fatal cases, where reporters may be more likely to err on the side of reporting due to the seriousness of the reaction. Other factors would need to be considered: co-morbidities, co-medications, genetics, and others. Therefore, some of these suspected ADRs may be due to third factor variables. Therefore, conclusions on the safety and risks of medicines cannot be made on the information obtained from the Drug Analysis Profiles alone. However, such data can be useful for hypothesis generation as in this study, standardized to prescribing rates, and supported by primary literature reports and case studies.
The available data on the polypharmacology of SGLT2i is incomplete. 86,87 For instance, there is also a lack of information regarding the inhibition of SGLT1 at various organ levels (except the kidneys and intestine) and inhibition of other SGLT transporters which may have unintended consequences. In this work, we have mitigated this risk by performing data integration of the pharmacological effects of SGLT2i integrating public databases, literature, and NDA documentation. However, not all SGLT2i had been comprehensively tested against all the targets we identified and their interactions with other target families remain unknown.

| CON CLUS ION
The results demonstrate a correlation between the selectivity of SGLT2i in relation to their respective suspected incidence of ADRs.
As a class, SGLT2i are a safe and effective medication for the treatment of T2DM with relatively low incidence of ADRs and fatalities and may have potential for the treatment for other diseases (cardioand nephroprotective properties).
Empagliflozin emerged as the most prescribed SGLT2i in the United Kingdom, the most selective SGLT2i on the market, and has the lowest incidence rate of suspected ADRs across the SGLT2i class. For established ADRs, empagliflozin had both a relatively low incidence for all ADRs (44.07), infections (4.61), amputations (0.53), and diabetic ketoacidosis (9.62) per 100,000 R x . Although a statistically significant difference between the SGLT2i could not be determined, empagliflozin consistently had the lowest ADR incidence out of all the studied SGLT2i.
Due to the possible linkage between SGLT2i selectivity and incidence of suspected spontaneous ADRs, we would predict that ertugliflozin will have a similar ADR profile as empagliflozin (as data emerge), because of the drug's high selectivity (≅2200-fold for SGLT2 over SGLT1).

ACK N OWLED G EM ENTS
The authors thank the ChEMBL, Yellow Card, and OpenPrescribing databases for open-source data.

D I SCLOS U R E
No conflict of interest to report.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in the supplementary material of this article.