Preclinical Data

An excellent overview of the biology of CD26/DPP-4 and its actions in the immune system with a focus on chemokines is found in Immunology Today 1999 20:367-375. Originally identified as a lymphocyte cell surface ADA binding protein with co-stimulatory activity, CD26 expression and activity are increased following T cell activation, and distinct subpopulations of CD26 bright T cells have been identified that subserve multiple functions including antigen recall, immunoglobulin synthesis, and activation of cytotoxic T cells CD26: a multifunctional integral membrane and secreted protein of activated lymphocytes. Scand J Immunol. 2001 Sep;54(3):249-64. Review.  

CD26 associates with other lymphocyte cell surface molecules including the chemokine receptor CXCR4, ADA and CD45, and monoclonal antibodies against CD26 promote aggregation of both CD26 and CD45 into lymphocyte lipid rafts CD26-mediated signaling for T cell activation occurs in lipid rafts through its association with CD45RO. Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):12138-43. Furthermore, CD26 directly binds to the cytoplasmic domain of CD45, providing a mechanism for engagement of specific signal transduction pathways leading to interleukin-2 production, a common downstream event secondary to CD26 activation .

Conversely, interleukin induces CD26 expression on a subset of human natural killer lymhocytes. Activation of lymphocyte CD26 leads to increases in intracellular calcium, tyrosine phosphorylation of multiple substrates, and cell proliferation. CD26 undergoes mannose-6 phosphorylation leading to interaction with the mannose 6- phosphate/insulin-like growth factor II receptor (M6P/IGFII) receptor following T cell activation. Soluble CD26 also interacts with the (M6P/IGFII) and enhances transendothelial T cell migration, an effect that requires its DPP-4 enzymatic activity Soluble CD26/dipeptidyl peptidase IV enhances transendothelial migration via its interaction with mannose 6-phosphate/insulin-like growth factor II receptor. Cell Immunol. 2002 Jan; 215(1):106-10.

What is the role of the catalytic domain of CD26 in the regulation of immune function? One approach to this issue involves the use of selective versus non-selective DPP-4 inhibitors for analysis of preclinical toxicity and effects on immune function. Lankas and colleagues examined the preclinical toxicity and effects on human T cells of a panel of selective (for DPP-4) vs. non-selective (cross-react against DPP-8/9, and QPP) inhibitors. Studies carried out in dogs demonstrated gastrointestinal toxicity of the DPP-8/9 inhibitors. Similarly, DPP-8/9 selective inhibitors produce considerable multi-system toxicity in rats and mice, resulting in mortality. The QPP-selective inhibitor produced reticulocytopneia in rats. Inhibitors of DPP-8/9, but not inhibitors of DPP-4, impaired human mononuclear cell proliferation following mitogen stimulation. See Dipeptidyl peptidase IV inhibition for the treatment of type 2 diabetes: potential importance of selectivity over dipeptidyl peptidases 8 and 9. Diabetes. 2005 Oct;54(10):2988-94

An alternative approach for assessing the biological importance of subdomains of CD26 is to employ variants of CD26 that have inactive catalytic domains or have lost the ability to bind the co-factor ADA. Yu and colleagues demonstrated using human lymphocytes in vitro that soluble CD26 is still capable of stimulating T lymphocyte proliferation independent of its catalytic or ADA binding activity Soluble CD26/Dipeptidyl Peptidase IV Enhances Human Lymphocyte Proliferation In Vitro Independent of Dipeptidyl Peptidase Enzyme Activity and Adenosine Deaminase Binding Scand J Immunol. 2011 Feb;73(2):102-11

The biological role(s) of DPP-8 and DPP-9 have not been extensively studied but they appear to exhibit enzymatic activity that overlaps with that described for DPP-4. Both enzymes cleave glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY, but with marked kinetic differences compared to dipeptidyl peptidase-4, as described in Dipeptidyl peptidase 8 and 9 specificity and molecular characterization compared to dipeptidyl peptidase IV. Biochem J. 2006 396:391-9

A link between CD26 expression/activity and levels of the proinflammatory chemokine stromal cell-derived factor-1 (SDF-1) was established in studies of experimental arthritis in mice, and observational studies in human subjects with rheumatoid arthritis. CD26-/- mice exhibited comparatively greater degrees of experimental joint inflammation following induction of antigen-induced arthritis, but no major differences in the basal humoral or cellular immune response were detected in CD26-/- vs control mice with AIA. However T cell activation led to increased interferon-g production in lymph node supernatants from CD26-/- mice. The cleavage of the chemokine SDF-1 was modulated by CD26 activity both in vitro, and in vivo. Intriguingly, the expression of the receptor for SDF-1, CXCR-4, was increased in CD26-/- joint tissue. These findings raise the possibility that CD26 expression may modify the activity of experimental inflammation in part through changing the levels of chemokines with inflammatory activity. See Circulating CD26 Is Negatively Associated with Inflammation in Human and Experimental Arthritis. Am J Pathol. 2005 Feb; 166 (2): 433-442

The majority of experiments assessing lymphocyte CD26 activity use specific antibodies for CD26 activation; whether the enzymatic peptidase activity of CD26 is involved in or required for multiple aspects of lymphocyte signaling has not always been conclusively determined as reviewed in CD26: an expanding role in immune regulation and cancer. Histol Histopathol. 2002 Oct;17 (4): 1213-26

Experiments carried out with mutant soluble CD26 molecules have demonstrated the importance of DPP-4 enzymatic activity for enhancement of T cell proliferation and induction of monocyte CD86 expression Soluble CD26/dipeptidyl peptidase IV induces T cell proliferation through CD86 up-regulation on APCs. J Immunol. 2001 Dec 15;167(12):6745-55

Similarly, anti-CD26 monoclonal antibodies inhibit T cell growth and proliferation via induction of G1/S arrest, effects which is also dependent on the enzymatic function of CD26 G1/S cell cycle arrest provoked in human T cells by antibody to CD26. Immunology. 2002 Nov;107(3):325-33.

Interpretation of data obtained from experiments using specific DPP-4 inhibitors to examine lymphocyte function is complicated by the specificity of the inhibitor employed, however DPP-4 inhibition has been shown to modify T and B cell proliferation, and cytokine production, as reviewed in CD26: a multifunctional integral membrane and secreted protein of activated lymphocytes. Scand J Immunol. 2001 Sep;54(3):249-64. Review.

The Fischer 344 mutant rat does exhibit defects in T cell subsets in response to immunization, or in the setting of experimental asthma. The number of CD4(+) T lymphocytes was markedly reduced compared to wild-type F344 and the decrease in T cell recruitment in CD26-deficient rats was associated with significantly reduced OVA-specific IgE-titres. See CD26 (dipeptidyl-peptidase IV)-dependent recruitment of T cells in a rat asthma model. Clin Exp Immunol. 2005 Jan;139(1):17-24

Yan and colleagues examined lymphocyte subpopulations, and the response to pokeweed mitogen (PWM) in CD26-/- mice. Several changes were observed in the cytokine response to PWM. Furthermore, serum levels of total IgG, IgG1, IgG2a and IgE, were lower in sera of CD26(-/-) mice following PWM and they detected reduced IL-4, IL-2 and delayed IFN-gamma production in the CD26-/- mice after PWM challenge. The authors conclude that CD26 contributes to the regulation of development, maturation and migration of CD4(+) T, NK and NKT cells, cytokine secretion, T cell-dependent antibody production and immunoglobulin isotype switching of B cells, as outlined in Deficiency of CD26 results in a change of cytokine and immunoglobulin secretion after stimulation by pokeweed mitogen. Eur J Immunol. 2003 Jun;33(6) :1519-27.

The available evidence suggests that the enzymatic activity of DPP-4 may not be essential for many of the T cell activating or co-stimulatory properties attributed to CD26, however not all experiments have used both wildtype and mutant CD26 molecules to examine this specific question. Similar questions have been raised about the role of CD26 in the brain. Moreover recent data examining the neurobiology of mutant Fischer 344 rats revealed unexpected changes in several parameters relating to CNS function including reduced body weight, reduced water consumption, increased pain sensitivity in a non-habituated hot plate test, and a reduced stress-like responses in tasks like the open field (OF), social interaction (SI), and passive avoidance test were found. See Extreme reduction of dipeptidyl peptidase IV activity in F344 rat substrains is associated with various behavioral differences. Physiol Behav. 2003 Oct;80(1):123-34.

Analysis of the immune response in CD26-/- (Dpp4-/-) mice provides direct evidence for an essential role of CD26 in the control of Th1 immune responses, via TGF-beta-1-dependent regulation of inflammation. CD26-/- mice exhibit diminished production of TGF-b-1 in response to antigenic stimulation, and markedly enhanced severity of experimental autoimmune encephalomyelitis (EAE). Furthermore, CD26-/- T cells exhibited enhanced proliferation and increased secretion of Th1-dependent cytokines such as IFN-g, IL-2, and TNF-a following antigenic stimulation. See TGF-beta1-Mediated Control of Central Nervous System Inflammation and Autoimmunity through the Inhibitory Receptor CD26. J Immunol. 2007 Apr 1;178(7):4632-40

In contrast, somewhat different results were obtained by Vora et al who examined the importance of DPP-4 activity or the entire DPP-4 molecule for T cell-dependent immune responses in a series of studies that used a) Dpp4-/- (CD26-/-) mice and b) the selective DPP-4 inhibitor, des-fluoro-sitagliptin. Dpp4-/- mice exhibit no cell surface expression of DPP-4, and no DPP-4 enzyme activity in plasma, yet had no significant differences in the numbers of leukocyte populations in blood, spleen or thymus. T cell proliferation, IL-2 production, and CD8 T cell function assessed by evaluation of cytotoxicity using a CTL assay was normal in Dpp4-/- mice. The humoral response to immunization with NP-CGG and NP-Ova was comparable in Dpp4-/- vs. Dpp4+/+ mice indicaitve of intact antigen-specific T cell-dependent antibody responses despite complete elimination of functional DPP-4. Splenic germinal center reactions, evaluated by the number and cellular quality of the GC reactions, were comparable in Dpp4+/+ vs. Dpp4-/- mice. Similarly, the ability to produce high affinity antibodies was not impaired following treatment of WT mice with des-fluoro-sitagliptin. As the Adenoseine deaminase (ADA) protein binds human, but not murine DPP-4, the biological results obtained in mice cannot be completely extrapolated to human subjects Genetic ablation or pharmacological blockade of dipeptidyl peptidase IV does not impact T cell-dependent immune responses BMC Immunol. 2009 Apr 9;10:19

The effects of sitagliptin on the immune system have been examined by Kim and colleagues in the NOD mouse model of type 1 diabetes. These experiments were prompted by the intriguing observation that sitagliptin reduced the development of type 1 diabetes in the NOD mouse Dipeptidyl peptidase IV inhibition with MK0431 improves islet graft survival in diabetic NOD mice partially via T-cell modulation Diabetes. 2009 Mar;58(3):641-51. Sitagliptin treatment of mice reduced the migration of CD4+ lymphocytes from spleen and lymph nodes in vitro. Differential labelling of CD4+ cells responsive or non-responsive to soluble CD26 in vitro following re-injection back into mice revealed preferential pancreatic homing for splenic CD4+ T cells and for incretin non-responsive LN CD4+ T cells. In contrast, soluble DPP4 increased migration of splenic but not thymic or lymph node lymphocytes. These effects required the catalytic activity of DPP-4, as they were abolished by co-treatment with sitagliptin. Moreover, direct incubation of lymph node cells with incretins differentially decreased lymph node (LN) lymphocyte migration. These findings were complemented by the demonstration that sDPP-4 activated Rac1 and increased VASP posphorylation on serine 157 in splenic CD4+ lymphocytes, actions mimicked by forskolin, and findings requiring the catalytic activity of DPP-4. In contrast, sDPP-4, GLP-1 and GIP had no effect on Rac1 activity or VASP phosphorylation in thymic or mesenteric LN preparations. GLP-1 and GIP increased phosphorylation of IkB, NFkB p65, and IKKa/b and increased nuclear localization and DNA binding of NFkB in LN CD4+ T lymphocytes, findings that were not mimicked by sDPP-4 or sitagliptin, nor evident in splenic or thymic CD4+ cells. Both GIP and GLP-1 reduced LN CD4+ T cell migration in a NFkB-dependent manner. Taken together, these findings contrast the differential actions of DPP-4 inhibitors on lymphocytes achieved through regulation of CD26, and via control of levels of intact incretin hormones and illustrate complexity in lymphocyte populations and their selective responsivity to CD26 and incretin hormones. Sitagliptin (MK0431) inhibition of dipeptidyl peptidase IV (DPP-IV) decreases NOD mouse CD4+ T cell migration through incretin-dependent and -independent pathways Diabetes. 2010 Jul;59(7):1739-50

Tian and colleagues carried out related studies examining the efficacy of the DPP-4 inhibitor NVP-DPP728 in NOD mice for 2-6 weeks. Diabetes was reversed in 57-73% of the diabetic NOD mice, with a modest reduction in insulitis score and increased circulating levels of TGF-b1, reduced insulitis, and increased numbers of thymic and pancreatic lymph node regulatory T cells. Mice with remission also exhibited increased circulating levels of TGF-b1 Reversal of New-Onset Diabetes through Modulating Inflammation and Stimulating {beta}-Cell Replication in Nonobese Diabetic Mice by a Dipeptidyl Peptidase IV Inhibitor. Endocrinology. 2010 May 5. [Epub ahead of print]

The DPP-4 inhibitor alogliptin markedly and rapidly attenuated LPS-induced Erk1/2 phosphorylation in U937 histiocyte-like cells in vitro. Alogliptin also reduced MMP-1 secretion, in the presence or absence of LPS, in some but not all experiments, depending on the culture conditions and stimulators of MMP-1 secretion. Alogliptin, in the absence of LPS (a ligand for toll-like recetor-4) also inhibited U937 cell proliferation, in some but not all experiments. Alogliptin also inhibited the LPS-stimulation of MMP-9, -12, and 15 gene expression but had no effect on TIMP expression in U937 cells. See DPP-4 (CD26) inhibitor alogliptin inhibits TLR4-mediated ERK activation and ERK-dependent MMP-1 expression by U937 histiocytes Atherosclerosis. 2010 Dec;213(2):429-35

Dobrian and colleagues treated high fat fed C57BL/6 mice with sitagliptin for 12 weeks. Sitagliptin reduced body weight, improved glycemia, improved GSIS in isolated islets, increased the number of small adipocytes and reduced adipose tissue Mac-2+ macrophage infiltration and decreased inflammation, principally cytokine gene expression (MCP-1, IL-12, IL-6, TNF-a, and IP-10), in adipose tissue and pancreatic islets. Whether these actions were due to glucoregulatory actions or weight loss or selective DPP-4 inhibition was not discerned. See Dipeptidyl peptidase-4 inhibitor sitagliptin reduces local inflammation in adipose tissue and in pancreatic islets of obese mice Am J Physiol Endocrinol Metab. 2010 Nov 16.

Shirakawa examined the consequences of DPP-4 inhibition on adipose tissue and liver, with a focus on immune cell infiltration and chemokine/cytokine expression, using haploinsufficient mice with beta cell inactivation of glucokinase (Gck+/- mice). Mice fed energy-enriched diets developed hypertrophy of adipose tissue, with increased CD11c+ proinflammatory macrophage infiltration and enhanced local expression of PAI-1 in adipocytes. Adipocyte stromal vascular fractions contained an increased proportion of F4/80+ adipose tissue macrophages in Gck+/- mice fed energy rich diets; Treatment of Gck+/- mice with des-fluoro sitagliptin signifcantly ameliorated the extent of adipocyte hypertrophy and inflammation and prevented the recruitment of M1 macrophages to visceral fat, in association with reduced expression of macrophage- and adipocyte-associated PAI-1 in adipocyte tissue. Neither des-fluoro sitagliptin or exendin-4 affected cytokine production from CD4+ or CD8+ T cells from mouse adipose tissue. des-fluoro sitagliptin also protected against the developed of hepatic steatosis in sucrose-fed mice Diet-Induced Adipose Tissue Inflammation and Liver Steatosis Are Prevented by DPP-4 Inhibition in Diabetic Mice Diabetes. 2011 Feb 17. [Epub ahead of print]

A compelling link between DPP-4 activity, CXCL10-dependent lymphocyte trafficking, and potentiation of immune-mediated cytoxicity has been provided in a series of preclinical studies by da Silva and colleagues Dipeptidylpeptidase 4 inhibition enhances lymphocyte trafficking, improving both naturally occurring tumorimmunity and immunotherapy Nat Immunol. 2015 Aug;16(8):850-8. Using sitagliptin to prevent degradation of CXCL10, or Dpp4-/- mice, and independent tumor engraft models, compelling evidence was provided that DPP4 inhibition potentiates a CXCL10-CXCR3 axis that enhances lymphocyte migartion into tumors, and enhances cytotoxiity, either alone, or more markedlty when combined with adjuvant-based tumor immunotherapies.

 

Clinical Data

A 24 week randomized placebo controlled study examined the consequences of sitagliptin administration on immune parameters in non-diabetic HIV+ subjects. Parameters examined included numbers of CD4+ T cells, levels of plasma HIV RNA, were measured every 4 weeks; fasting levels of RANTES, stromal derived factor (SDF)-1α, and Soluble TNF receptor II. Sitagliptin had no effect on the majority of parameters assessed, whereas levels of SDF-1a declined in sitagliptin-treated patients. Dipeptidyl Peptidase IV Inhibition Does Not Adversely Affect Immune or Virological Status in HIV Infected Men And Women: A Pilot Safety Study J Clin Endocrinol Metab. 2012 Dec 21

A second study in HIV+ subjects examined the effects of sitagliptin or placebo, administered 100 mg daily for 8 weeks in male and female subjects treated with anti-retroviral therapy and impaired glucose tolerance. Plasma levels of HIV RNA remained undetectable in both groups throughout the trial. Sitagliptin therapy reduced circulating levels of CRP, and CXCL10, reduced adipose tissue abundance of MCP-1 mRNA, and increased circulating endothelial progenitor cell numbers. Individual changes in levels of inflammatory markers did not corelate with improvements in glucose homeostasis. Sitagliptin Reduces Inflammation and Chronic Immune Cell Activation in HIV+ Adults with Impaired Glucose Tolerance J Clin Endocrinol Metab. 2015 May 4:jc20151531

Price and colleagues assessed the effects of 4 weeks of sitagliptin treatment on parameters of immune function in healthy non-diabetic subjects using a randmized controlled trial design. 41 healthy subjects (3:1 randomization, sitagliptin vs. placebo) were studied. The primary study outcome was the change in levels of TGF-beta protein in plasma at day 28. The percent DPP-4 inhibition (measured in fasting subjects at drug trough) was ~50-60%. Plasma levels of TGF-beta were stable and did not change over time in either placebo-or sitagliptin-treated groups. Similarly, no changes in (a large panel of) levels of cytokines and chemokines (including known DPP-4 substrates such as RANTES and IP10), regulatory T cells, or WBCs, platelets or neutrophils were observed after exposure to sitagliptin. Changes in circulating immune subsets consistent with induction of CD26 expression were observed after sitagliptin. Gene expression in RNA isolated from whole blood identified small changes in expression of several mRNA transcripts encoding proteins that might control cell trafficking. LPS stimulation followed by analysis of cytokine production or analysis of stimulated T cell proliferation ex vivo did not reveal significant differences between cells isolated from subjects treated with placebo or sitagliptinEffects of Short-Term Sitagliptin Treatment on Immune Parameters in Healthy Individuals, A Randomized Placebo-Controlled Study. Clin Exp Immunol. 2013 May 27. doi: 10.1111/cei.12144

Anz and colleagues incubated human peripheral blood mononuclear cells (PBMCs) with either sitagliptin, vildagliptin, or saxagliptin in vitro, and observed no effect on cytokine production, or upregulation of CD80, CD86, or MHC-II following stimulation  with the TLR7 ligand R84. Similarly, induction of C69, T cell proliferation and IFN-g secretion were not affected by DPP-4 inhibition. Mice treated with vildagliptin normal T cell activation or lymphocyte migration

The dipeptidylpeptidase-IV inhibitors sitagliptin, vildagliptin and saxagliptin do not impair innate and adaptive immune responses Diabetes Obes Metab. 2013 Dec 5. doi: 10.1111/dom.12246

Makdissi and colleagues examined the mRNA expression of chemokines and inflammatory cytokines in white blood cells from patients with T2DM treated with sitagliptin for 12 weeks or in subjects after the first dose of sitagliptin. All samples were collected in the fasting state. Sitagliptin administration was associated with reduced expression of proinflammatory molecules, and in some instances, significant reductions were evident within 2 hrs after the first dose of sitagliptin. Plasma levels of CRP and IL-6 also were significantly reduced after sitagliptin treatment. Sitagliptin Exerts an Antinflammatory Action J Clin Endocrinol Metab. 2012 Jun 28.

Yamauchi and colleagues reported two patients who developed remitting seronegative symmetrical synovitis with pitting edema (RS3PE), one while taking sitagliptin (5 weeks of exposure), the other while taking vildagliptin (8 weeks of exposure). Discontinuation of sitagliptin led to marked clinical improvement after 7 days, however predinsone was required for reslution of the cutaneous inflammation. After cessation of vildagliptin the signs and symptoms of RS3PE resolved within 10 days. RS3PE in Association With DipeptidylPeptidase-4 Inhibitor: Report of Two Cases Diabetes Care February 2012 35:e7; doi:10.2337/dc11-1995

 

Skandalis and colleagues reported 5 patients with type 2 diabetes taking metformin and either vildagliptin(4) or sitagliptin (1) who developed bullous pemphigoid. Patients were taking the DPP-4 inhibitors for 2-13 months prior to the diagnosis of BP. The cluster of cases, the response to therapy following the discontinuation of the DPP-4 inhibitors, and the lack of previous association between BP and T2DM raises the possibility of a cause-effect relationship, although rechallenge was contraindicated in view of the potential life-threatening severity of severe BP. The authors provide a range of mechanistic hypotheses for their observations. See Drug-induced bullous pemphigoid in diabetes mellitus patients receiving dipeptidyl peptidase-IV inhibitors plus metformin J Eur Acad Dermatol Venereol. 2011 Apr 6. doi: 10.1111/j.1468-3083.2011.04062.x. [Epub ahead of print]

Two additional cases of BP were described in Greek subjects treated with metformin, followed by addition of vildagliptin. The cases of BP developed 2 months after the initiation of vildagliptin therapy. Complete remission was achieved 8-10 weeks after discontinuation of vildagliptin Dipeptidyl peptidase-4 inhibitors cause bullous pemphigoid in diabetic patients: report of two cases Diabetes Care. 2011 Aug;34(8):e133

In contrast, Nishioka et al described marked improvement in psoriasis after initiation of sitagliptin, 50 mg once daily, for T2DM in a patient with a 17 year history of plaque psoriasis. Three months after starting sitagliptin, the psoriasis was markedly improved, independent of any changes in A1c. Sitagliptin, a Dipeptidyl Peptidase-IV Inhibitor, Improves Psoriasis Dermatology. 2011 Nov 1.