The success and ever increasing interest in incretin-based therapies and drugs especially sitagliptin, vildagliptin and exenatide depict their immense importance in improving patient outcomes in T2DM (Nasr and Sadek, 2022). Incretin effect is the enhancement in the secretion of insulin as a function of increase in oral intake of glucose. For a given rise in plasma glucose concentration, the increase in plasma insulin is roughly threefold times greater, if glucose is administered orally compared to intravenous route (Perley and Kipnis, 1967). The main incretin hormones secreted by the epithelial cells of the gastrointestinal track are glucagon-like peptide-1 (GLP)-1 and glucose-dependent insulinotropic peptide, also known as Gastric Inhibitory Polypeptide (GIP), which stimulate the pancreatic cells (α and β cells) and regulate insulin secretion with respect to blood glucose concentration (Drucker, 2006).

The first incretin to be reported was GIP. It is secreted in a single biologically active form by the K-cells in the duodenum and jejunum upon consumption of carbohydrates or lipids. Its main functions are stimulation of insulin secretion in a glucose dependent manner and contribution to fat metabolism in adipocytes. It also has proliferative effect on β-cells. The second important incretin hormone is GLP-1, released from L-cells in the distal ileum and colon (Baggio and Drucker, 2007; Gautier et al., 2008). GLP-1 is similar to but unlike GIP, actions of GLP-1 are better preserved in type 2 diabetes mellitus (T2DM) patients, which makes it a highly desirable target for drug discovery for T2DM (Sharma and Bhatia, 2020); (Nauck et al., 1993).

However, both GLP-1 and GIP are quickly degraded by dipeptidyl peptidase 4 (DPP-4) enzyme (Figure 1). It cleaves the oligo-peptides after the 2nd amino acid from the N terminal proline or alanine sequences, thus rendering GLP-1 and GIP inactive (Deacon et al., 1995). In addition to that, DPP-4 enzyme also deactivates more than 40 physiologically active hormones, neuropeptides, cytokines, and other proteins in vivo through dipeptide cleavage (Mentlein et al., 1993). This very short half-life (<2 min) of GLP-1 due to the action of DPP-4 is a formidable challenge in the incretin-based drug development for T2DM.

DPP-4 is member of a family of proteases which also includes dipeptidyl-peptidase-8 (DPP-8), dipeptidylpeptidase-9 (DPP-9) and fibroblast activation protein (FAP) (Ajami et al., 2004). DPP-4 enzyme is a 766 amino acid trans membrane glycoprotein, also known as adenosine deaminase complexing protein 2 or CD26 (Jose and Inzucchi, 2012). It is a serine amino peptidase enzyme, which exists on the superficial side of epithelial and endothelial cells (involving monocytes and lymphocytes) and propagates in plasma (Fadini and Avogaro, 2011). These enzymes are broadly distributed in various tissues (brain, lungs, kidneys), T-cell, B-cell and natural killer cells (Lambeir et al., 2003). DPP-4 has two mechanisms of action: first as a membrane spanning protein, where it binds and activates adenosine deaminase complexing protein and transfers intracellular signals via dimerization; and second as an enzyme (Lambeir et al., 2003). Thus, a DPP-4 can circulate freely or can exist in the membrane bound form.

As shown in Figure 4, the β-propeller domain is packing against the α/β hydrolase domain. In addition to that, at the interface of β-propeller domain and α/β hydrolase domain, catalytic triad (residues Ser⁶³⁰, His⁷⁴⁰ and Asp⁷⁰⁸) is located (Aertgeerts et al., 2004), with both these domains participating in the binding of the inhibitor. In the DPP4-like protein family, glutamic acid-rich loop (Glu²⁰⁵/Glu²⁰⁶) is highly conserved and is responsible for substrate recognition. The catalytic serine S⁶³⁰ falls within the pentapeptide sequence Gly⁶²⁸-X-Ser⁶³⁰-Tyr⁶³¹-Gly⁶³², which is highly conserved across the α/β hydrolase protein family and is indispensable for catalytic activity.

Studying the binding interactions of DPP-4 inhibitors at the binding site are crucial in understanding their performance and also for guiding research into novel drug candidates. The DPP-4 binding site can be divided into two pockets viz. S1 and S2 and a hydrophobic sub-S1 or sub-S3 sub-pocket. Figure 5 represents a general DPP-4 ligand. It contains an aromatic group, a heterocycle, a primary amine, as well as a nitrile group. S1 pocket is usually occupied by the aromatic ring or the saturated heterocycle, whereas the primary amine group forms hydrogen bond with Glu^205/206 and Tyr⁶⁶². S2 site binds with the aromatic heterocycle or fused rings present in the inhibitor (Metzler et al., 2008). The hydrophobic nature of the sub-pockets dictate that these are occupied by an aromatic group, and linker is necessary between the S1 and sub-S1 binding ligands (Deng et al., 2018). A common feature amongst most DPP-4 inhibitors approved by regulatory authorities is the presence of a cyanopyrrolidine moiety. The presence of the nitrile group allows the ligand to catalytically bind to the active serine hydroxyl group (Kumar et al., 2021).

Their main interaction is with the DPP-4 complex which are recognized for competitive inhibition (Jose and Inzucchi, 2012). DPP-4 enzyme inhibition also regulates the action of various cardio active elements, neuropeptide Y and stromal cell derived factor-1 (SDF-1) (Drucker, 2007). Fibroblast activation protein, DPP-2, DPP-8 and DPP-9 show DPP-4 enzyme like activity and therefore DPP-4 inhibitors must be selective (Green et al., 2006).

DPP-4 inhibitors are already being employed in T2DM treatment globally. Table 2 lists all the DPP-4 inhibitors till date in various stages of drug development (Kumar et al., 2021).

DPP-4 inhibitors are characterized into three different classes based on their binding sites (refer to Figure 5 from Section 2) (Tomovic et al., 2018). Vildagliptin and saxagliptin make up Class 1 where binding occurs only on the S1 and S2 binding sites between the nitrile group of the cyanopyrrolidine group and Ser⁶³⁰ of the active site on DPP-4. Saxagliptin is five times more active in blocking DPP4 than vildagliptin. Class 2 is composed of linagliptin and alogliptin where binding interaction with the S1 sub pocket or in case of the former with S2 sub pocket as well, giving it eightfold higher activity than alogliptin. The uracil rings in both ligands force a conformational alteration of Tyr⁵⁴⁷ within the S2 sub pocket. Class 3 consists of ligands binding with the S2-extensive site. Sitagliptin and tenegliptin form this class which has the highest inhibitory capability among gliptins (Röhrborn et al., 2015).

Sitagliptin, alogliptin and linagliptin possess high selectivity for DPP-4 due to strong binding with S2-extensive site which is absent in related peptidases like DPP-8, DPP-9, and FAP, wherein vildagliptin seems less selective. These inhibitors are appropriate for once or twice daily dosage, orally active are absorbed relatively fast. They are all excreted renally after negligible metabolism, except linagliptin which is eliminated via the biliary pathway (Deacon, 2019; Gallwitz, 2019). Insulin secretion can be enhanced by sulphonylureas independently and therefore introduce diabetic patients to a heightened risk of hypoglycaemia. DPP-4 inhibitors are thus added to the sub-maximal limit of sulphonylurea to achieve tighter glycaemic control (Gerich, 2010). In fact, DPP-4 inhibitors are now replacing the use of sulphonylureas as insulin-releasing agents due to their insulin-tropic effect, and notably because DPP-4 inhibitors reduce the intrinsic risk of low blood sugar or hypoglycaemia. Also, DPP-4 inhibitors are body weight neutral, in contrast to sulphonylurea therapy which is linked with body weight gain (Palmer et al., 2016). Differences between DPP-4 inhibitors with various classes of antidiabetic agents are shown in Table 3. Prior experiments in vitro have exhibited that DPP-4 inhibitors impede T-cell proliferation and cytokine expression and these agents have also been examined in animal models, showing potential anti-inflammatory effects (Yazbeck et al., 2009).

Sitagliptin was the first commercialized, orally active and selective DPP-4 inhibitor (Dhillon, 2010), which was capable of stimulating a 2-fold increase in post-meal active plasma GLP-1 levels with respect to placebo (Lim et al., 2009). It is presently available as a single agent or in fixed-dose association with metformin (Reynolds et al., 2008). A 50 mg daily dosage produces 80% inhibition of DPP-4 activity in healthy subjects and type 2 diabetic patients (Herman et al., 2006a). It is extremely selective for DPP-4 (upto 2600 times higher than other DPP family enzymes (Richter et al., 2008a). Additionally, sitagliptin improves surrogate markers of β-cell function in humans and enhanced β-cell mass in animal studies (Palalau et al., 2009).

Saxagliptin is an incredibly selective and reversible DPP-4 inhibitor (Tahrani et al., 2009a). It can increase the GLP-1 levels to 1.5 to 3-fold with a single dose of 2.5–400 mg which inhibits DPP-4 activity between 50% and 79% (Chacra, 2010). Saxagliptin is tenfold more effective than vildagliptin and sitagliptin. Its pharmacokinetics permit once-daily administration and are not influenced by age or gender. The pharmacokinetics of other drugs are also not affected by combination of saxagliptin with some other (oral antidiabetic agents) OADs such as metformin, pioglitazone, or glyburide in a remarkable way (Tahrani et al., 2009a). Saxagliptin improved β-cell function when employed as mono-therapy or in combination with metformin (Jadzinsky et al., 2009). However the similar improvement was not seen when combined with suboptimal glyburide (Chacra et al., 2009). The FDA endorsed saxagliptin for its use as a supplement to diet and exercise to improve glycaemic control in adults for curing type 2 diabetes (Anz et al., 2014). Saxagliptin can be employed in mono-therapy or in combination with other OADs including metformin, sulphonylureas or TZDs (Karyekar et al., 2011).

Vildagliptin is another selective DPP-4 inhibitor that can produce 1.5- to 3-fold increase in post meal active plasma GLP-1 levels vis-à-vis placebo (Hu et al., 2009). A 100-mg vildagliptin dose is enough to completely suppress the activity of DPP-4 in patients with T2DM (Balas et al., 2007). Vildagliptin also improved β-cell replacement function in humans and stimulated β-cell mass increase in animal studies (Mari et al., 2008). It improved HbA1c and reduced FPG and PPG in type 2 diabetic patients when used in mono-therapy or in combination with some other OADs. With vildagliptin the improvement of glycaemic control lasts at least 2 years (Scherbaum et al., 2008), and is better in patients with greater initial HbA1c levels (Rosenstock et al., 2007). Vildagliptin has also been identified to be effective in the older population (Das et al., 2021). Due to their compatible mechanisms of action and low risk of hypoglycaemia, vildagliptin and metformin have been formulated in a single tablet (Tahrani et al., 2009b).

Alogliptin is another effective, highly selective DPP-4 inhibitor approved in 2010. It can be employed in mono-therapy or in combination with metformin or glyburide for T2DM patients. The improvements in fasting glycaemia and HbA1c are maintained for at least 26 weeks. Alogliptin possesses good gastrointestinal tolerability, is weight neutral and has a very low degree of hypoglycaemia risk (Nauck et al., 2009). Administration of alogliptin with metformin, pioglitazone or glibenclamide in healthy patients suggests no significant drug-drug interactions (Karim et al., 2009). Alogliptin in combination with a specific dose of insulin, improved glycaemic control without elevating hypoglycaemia rates and without magnifying weight gain (Rosenstock et al., 2009a). Notably, greater reductions in HbA1c levels were identified when alogliptin was combined with pioglitazone, without elevating the possibility of hypoglycaemic events (Deacon, 2011). The therapeutic effectiveness of alogliptin-pioglitazone combination deserves attention in the future management of T2DM (Argyrakopoulou and Doupis, 2009).

Other important approved DPP-4 inhibitors as listed in Table 2 are Linagliptin, Gemigliptin L-tartrate sesquihydrate, Anagliptin, Tenegliptin Hydrobromide Hydrate, Denagliptin tosilate, Omarigliptin, Evogliptin Hydrochloride and Trelagliptin Succinate.

As can be seen from Figure 6, DPP-4 inhibitors constitute a heterogeneous class of small molecules with diversity and variations in chemistry, in pharmacokinetic properties such as absorption, distribution, metabolism and excretion routes, and in pharmacodynamic components such as potency and selectivity of DPP-4 inhibition (Scheen, 2012a). From a pharmacological point of view, tight-binding inhibitors are crucial as once they are bound to their target, inhibition of the enzyme operation continues even after the loose drug has been removed from circulation or from the vicinity of the site of action, a profile which is ideal for once-daily dosing. Currently available DPP-4 inhibitors display slow, tight-binding inhibition kinetics and are reversible competitive inhibitors of DPP-4 (Villhauer et al., 2003).

Despite clinical dissimilarities within the group, all DPP-4 inhibitors have a common working action. Firstly, all DPP-4 inhibitors possess pronounced structural heterogeneity which may account for differences in the pharmacokinetic properties. Linagliptin, for instance, possesses distinctive xanthine-based structure (Deacon and Holst, 2010). Its terminal half-life of 184 h (Hüttner et al., 2008), is substantially longer than that of sitagliptin (10–12 h) (Neumiller, 2009). Saxagliptin assimilation by CYP3A4/5 produces an active metabolite, however metabolites of linagliptin, vildagliptin and sitagliptin seem to be inactive. Also, terminal half-life of saxagliptin is 2.5 h, notably less than the mean half-life of 26.9 h for plasma DPP-4 inhibitors. A direct consequence is that if patients miss a dose, the long terminal half-life of linagliptin would predictably retain effectual glycaemic control whereas the short half-lives of sitagliptin and saxagliptin indicate that patients need to strictly follow the dosing interval (Gerich, 2010). When handling T2DM patients with renal dysfunction, the differences in the path of metabolism of action may also prove pivotal. Linagliptin is unlikely to accrue in renally impaired T2DM patients unlike other DPP-4 inhibitors that are removed renally. Due to the strong binding capacity of linagliptin with DPP-4, it rapidly saturates at low doses (Fuchs et al., 2009) and therefore any free linagliptin not bound to DPP-4 is quickly eliminated (Ahmad, 2007). Linagliptin is primarily excreted unchanged by enterohepatic mechanism and thus is appropriate in renally impaired patients. Vildagliptin, however, is excreted via the kidney by instant breakdown into an active metabolite, with an absolute bioavailability being 85% (He, 2012).

DPP-4 inhibitors exhibit appreciable safety and tolerance profile. Some recurrent adverse effects are reported such as nasopharyngitis and skin lesions but these are not serious enough to lead to discontinuation of the treatment.

The effectiveness and safety profile of these inhibitors manifests a favourable scenario particularly for elderly subjects, but only when any influence on complications is not needed (Gallwitz, 2016; Sesti et al., 2019). Other side effects include infection in upper respiratory tract, urinary tract infection and headache. Urticarial dermatological reactions and angioedema have also been stated. Also, T2DM patients face twice the risk of acute pancreatitis and various retroactive studies and meta-analysis indicate that DPP-4 inhibitor therapy actually lowers said risk (Pinto et al., 2018). These safety profile of DPP-4 inhibitors is summarized in Figure 7.

DPP-4 has consequences beyond its proteolytic action which includes T-cell activation and proliferation (Lambeir et al., 2003). DPP-4 inhibitors may prolong the action of various hormones. The potential side-effects associated with the action of these hormones include neurogenic inflammation, elevated blood pressure and magnified general inflammation and allergic reactions (chemokines). Such side-effects have not been observed in preclinical or clinical research yet.

Linagliptin (Forst et al., 2010), saxagliptin (Rosenstock et al., 2009b), vildagliptin (Pi-Sunyer et al., 2007), sitagliptin (Aschner et al., 2006), and alogliptin (DeFronzo et al., 2008b) shows mild tolerability with adverse event profiles compared to placebo. Currently, linagliptin is the only DPP-4 inhibitor that showed improvement in wound healing in preclinical studies (Gupta and Kalra, 2011). The wide distribution of DPP-4 in various tissues has led to concern that they may have increased risk of infectious and inflammatory processes. DPP-4 adjusts levels of various mediators required in immune modulation and a meta-analysis report identifies elevated risk of infections with sitagliptin, but not with vildagliptin (Richter et al., 2009). A systematic review disclosed that DPP-4 inhibitors slightly increase the risk of nasopharyngitis and urinary tract infections in contrast with non–incretin-based placebo or OAD (Amori et al., 2007). Nasopharyngitis is detected in ≥5% of sitagliptin cured patients (Fasanya, 2021).

Utilising DPP-4 inhibitors in clinical practice is comparatively easy than other OADs as there is no need of dose titration and adjustment when used with other prescribed medications. In monotherapy, they could challenge traditional OADs. Saxagliptin is associated with appreciable drug–drug interactions, whereas both sitagliptin and vildagliptin demonstrate minor inclination for drug–drug interactions. As DPP-4 inhibition mainly supports physiological roles of endogenous GLP-1, these inhibitors may be of significant relevance in curing T2DM or even pre-diabetes, but this remains to be confirmed in comprehensive clinical trials. A review of Cochrane mentions the evaluation of sitagliptin and vildagliptin over a period of 12 and 52 weeks. The report disclosed lowering of HbA1c levels approximately 0.7% and 0.6%, respectively, in contrast to placebo (Richter et al., 2008b). Monami et al. carried out a meta-analysis comparing the surplus glycaemic effects of DPP-4 inhibitors and numerous anti-hyperglycaemic agents on the lipid profile in T2DM patients. It was observed that DPP-4 inhibitors were slightly more effective in lowering total cholesterol and triglycerides vis-à-vis other agents (Monami et al., 2012). DPP-4 inhibitors have a unique action pathway vis-à-vis other existing class of glucose lowering agents taken orally (Krentz et al., 2008). They enhance β-cell proliferation and neogenesis and also inhibit apoptosis (Butler et al., 2003). Apart from mono-therapy, DPP-4 inhibitors have shown promise in several combinations with other glucose-lowering agents (Neumiller et al., 2010). Combination of DPP-4 inhibitors with existing drugs could be a welcome supplement. The most favoured combination of DPP-4 inhibitors is with metformin (Dahut and Madan, 2010). Combining metformin/DPP-4 inhibitor resulted in higher GLP-1 concentrations compared to DPP-4 inhibitor mono-therapy (Vardarli et al., 2014). Rather in one of the study, metformin itself acts as a DPP-4 inhibitor by inhibiting the DPP-4 enzyme in the fasting state (Cuthbertson et al., 2009). Moreover, with this dual therapy (metformin/DPP-4 inhibitor), patients can also receive additional treatment intensification with sodium-glucose cotransporter-2 (SGLT-2) inhibitor or thiazolidinedione (TZD) (Inzucchi et al., 2015). Other combinations include sulphonylurea, thiazolidinedione or metformin and sulphonylurea combined therapy (Drucker and Nauck, 2006).

Combination of GLP-1RAs with DPP-4 inhibitors is not advisable, because no clinically significant auxiliary benefit is predicted, although there are no concerns about adverse effects (Davies et al., 2018); (American Diabetes Association, 2019). Patients unable to tolerate GLP-1RAs treatment are recommended for DPP-4 inhibitor therapy. Also, it is advised that DPP-4 inhibitor therapy should cease when treatment is taken forward with an injectable (GLP-1RAs) therapy, at later stages (American Diabetes Association, 2019).

New incretin-based therapies provide numerous options for glycaemic control in patients with T2DM and have clear advantages over other classical glucose-lowering agents (Phung et al., 2010), yet estimated cost of the therapy is an essential factor when making global comparisons for clinical usage (Waugh et al., 2010). DPP-4 inhibitors undoubtedly are more expensive than sulphonylureas, but more affordable than GLP-1 receptor agonists. At the moment, only sparse preliminary data are available (Schwarz et al., 2008). Thus, further economic analyses are necessary to support the switch from sulphonylureas to DPP-4 inhibitors (Waugh et al., 2010). In addition to this, further studies are needed to explore the long-term safety and efficacy of DPP-4 inhibitors. This will help establish their role in the management of T2DM and provide clinicians with valuable information to make informed treatment decisions. Additionally, identifying patient populations that are most likely to benefit from DPP-4 inhibitor therapy is crucial for personalized treatment plans. This can be achieved through ongoing clinical trials and real-world evidence studies. Furthermore, research is needed to investigate the potential benefits of combination therapy with DPP-4 inhibitors and other glucose-lowering agents. This could help improve overall glycemic control and reduce the risk of adverse effects associated with higher doses of single agents. Overall, ongoing research in the field of DPP-4 inhibitors holds promise for improved management of T2DM and better patient outcomes.

Leveraging incretin effect for treating T2DM is a relatively novel line of treatment. DPP-4 inhibitors are rapidly catching attention as they not only complement but also extend the traditional therapeutic avenues to treat T2DM. Improvement in T2DM in terms of delay in gastric emptying, reduction in postprandial glucose levels, and inhibition of glucagon secretion is observed with DPP-4 inhibitors. As recounted above, they can be efficiently used in combination therapy. Moreover, they have the advantage of meeting medical needs in metabolic disorders, which is especially significant for elderly diabetic patients or patients having obesity problems along with T2DM. In addition to that, evidence increasingly points towards reversal of dysfunction of pancreatic β cells. It is believed that the anti-apoptotic and proliferative characteristics of DPP-4 inhibitors can help in the regeneration of pancreatic β cells. Research also needs to be focused on finding the correct intervention point to mitigate progressive loss of βcells mass and function—the hallmark of prediabetes.

Though many questions have been raised regarding the safety profile of DPP-4 inhibitors particularly in areas of pancreatitis and pancreatic cancer, not much evidence is available for it. One should be cautious nonetheless and further research is needed in this regard. GLP-1 stimulation, at this stage should prove beneficial but again little evidence exists to support this hypothesis and more studies are needed for clarification in this regard.

The most disappointing result is its negligible favourable effect on cardiovascular and renal outcomes compared to other new antidiabetic agents like SGLT-2 inhibitors or GLP-1 RAs.

Apart from the cyanopyrollidine scaffolds mentioned above, research groups and pharmaceutical companies all over the world are developing multiple new candidates, many of which are already undergoing clinical trials. This is testament to the importance of harnessing the incretin effect as an attractive approach for T2DM therapy, as well as the role of DPP-4 as a drug target in achieving this goal.

The best treatment option for diabetes would not only control blood sugar levels but would also help in managing overall risk factors. In this context, DPP4 inhibitors seem to be very good candidates as are not known to cause hypoglycemia or weight gain, no requirement of dose escalation and have a favorable anti-inflammatory and safety profiles. Also, they are relatively safer for elderly diabetic patients and kidney disease patients with diabetes. They have been used quite extensively and thus there is lot of experience in their use. All these factors make them excellent choices and it can be safely said that all the hype surrounding DPP-4 inhibitors is very well justified.