Recent advances and structure-activity relationship studies of DPP-4 inhibitors as anti-diabetic agents.

Diabetes mellitus (DM) is one of the largest public health problems worldwide and in the last decades various therapeutic targets have been investigated. For the treatment of type-2 DM (T2DM), dipeptidyl peptidase-4 (DPP-4) is one of the well reported target and has established safety in terms of cardiovascular complexicity. Preclinical and clinical studies using DPP-4 inhibitors have demonstrated its safety and effectiveness and have lesser risk of associated hypoglycaemic effect making it suitable for elderly patients. FDA has approved a number of structurally diverse DPP-4 inhibitors for clinical use. The present manuscript aims to focus on the well reported hybrid and non-hybrid analogues and their structural activity relationship (SAR) studies. It aims to provide structural insights for this class of compounds pertaining to favourable applicability of selective DPP-4 inhibitors in the treatment of T2DM.

Tetrazoles and Related Heterocycles as Promising Synthetic Antidiabetic Agents.

Tetrazole heterocycle is a promising scaffold in drug design, and it is incorporated into active pharmaceutical ingredients of medications of various actions: hypotensives, diuretics, antihistamines, antibiotics, analgesics, and others. This heterocyclic system is metabolically stable and easily participates in various intermolecular interactions with different biological targets through hydrogen bonding, conjugation, or van der Waals forces. In the present review, a systematic analysis of the activity of tetrazole derivatives against type 2 diabetes mellitus (T2DM) has been performed. As it was shown, the tetrazolyl moiety is a key fragment of many antidiabetic agents with different activities, including the following: peroxisome proliferator-activated receptors (PPARs) agonists, protein tyrosine phosphatase 1B (PTP1B) inhibitors, aldose reductase (AR) inhibitors, dipeptidyl peptidase-4 (DPP-4) inhibitors and glucagon-like peptide 1 (GLP-1) agonists, G protein-coupled receptor (GPCRs) agonists, glycogen phosphorylases (GP) Inhibitors, α-glycosidase (AG) Inhibitors, sodium glucose co-transporter (SGLT) inhibitors, fructose-1,6-bisphosphatase (FBPase) inhibitors, IkB kinase ε (IKKε) and TANK binding kinase 1 (TBK1) inhibitors, and 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). In many cases, the tetrazole-containing leader compounds markedly exceed the activity of medications already known and used in T2DM therapy, and some of them are undergoing clinical trials. In addition, tetrazole derivatives are very often used to act on diabetes-related targets or to treat post-diabetic disorders.

Transitioning from insulin to dipeptidyl-peptidase 4 (DPP-4) inhibitors for type 2 diabetes.

Insulin administration is time intensive and costly in facility staffing. When we started nursing home patients with type 2 diabetes (T2D) on DPP-4 inhibitors, we tapered insulin when finger stick blood sugar levels dropped to <200 mg/dL. Of 34 patients we were able to stop mealtime insulin in 28 (82%) and stop all insulin in 20 (59%). On average, hemoglobin A1c (HbA1c) decreased 0.5% and weight by 2.8 pounds. Among the 20 who stopped all insulin, HbA1c improved in 11 on average 1% (p=0.02), and weight decreased in 11 on average 4.1 pounds (p=0.66). 12 patients were switched in one day because of a low insulin dose or low HbA1c Tapering duration in the other 8 ranged from 10-727 days with an insulin dose of 28 to 84 units daily. Larger studies are needed to confirm our findings, develop a protocol for tapering insulin, and measure hypoglycemia, comfort and cost.

Angiogenic T cells are decreased in people with type 2 diabetes mellitus and recruited by the dipeptidyl peptidase-4 inhibitor Linagliptin: A subanalysis from a randomized, placebo-controlled trial (RELEASE study).

Angiogenic T (Tang) cells are mediators of vascular repair, and are characterized by surface expression of CXCR4. This receptor for stromal cell-derived factor-1α (SDF-1α) is cleaved by dipeptidyl peptidase-4 (DPP-4). Tang cell levels were investigated in people with type 2 diabetes mellitus (T2DM) compared with matched healthy controls and after treatment with the DPP-4 inhibitor Linagliptin. People with T2DM were randomized to 5 mg/day Linagliptin (n = 20) or placebo (n = 21) for 26 weeks. Tang cell frequency was identified in peripheral blood mononuclear cells (CD3+ CD31+ CXCR4+ ) and levels of endothelial progenitor cells (EPCs) (CD34+ CD133+ KDR+ ) were also assessed in whole blood. Circulating Tang cell levels were significantly lower in people with T2DM compared with the healthy control group. SDF-1α levels increased significantly in Linagliptin-treated people with T2DM compared to placebo, and a trend was observed in change of Tang cell levels, while EPC count did not change. In conclusion, circulating Tang cell levels were considerably lower in people with T2DM, while a trend was observed in recruitment of Tang cells after 26 weeks of treatment with Linagliptin. These data suggest that DPP-4 inhibitors may potentially exert beneficial effects on bone marrow-driven vascular repair.

Chronic kidney disease in type 2 diabetes: Implications for managing glycaemic control, cardiovascular and renal risk.

This review examines the current literature relating to diabetes related kidney disease (DKD) and the optimal management of cardio-renal risk. DKD develops in approximately 40% of patients with type 2 diabetes mellitus. The mainstay of therapy is to reduce the progression of DKD by optimising hyperglycaemia, blood pressure, lipids and lifestyle. Evidence supports the role for renin-angiotensin system blockade in limiting the progression of DKD. Recent data from diabetes related cardiovascular outcome trials and renal specific trials have provided a novel insight on the additional benefits of sodium-glucose cotransporter-2 inhibitors (SGLT2i) in reducing the progression of DKD as well as cardiovascular risk. Lessons have been learnt from CREDENCE and there are expectations that DAPA-CKD and EMPA-KIDNEY will further support the benefits of SGLT2 inhibition in relation to DKD. As a consequence, international guidelines have been updated to reflect the positive benefits. In addition, novel steroidal mineralocorticoid receptor antagonists offer a potential role in future years. The review examines the current evidence and future approach to optimising outcomes for renal protection in patients with diabetes.

Dipeptidyl peptidase 4 inhibitors vs. metformin for new-onset dementia: a propensity score-matched cohort study.

BACKGROUND: Hypoglycemic pharmacotherapy interventions for alleviating the risk of dementia remains controversial, particularly about dipeptidyl peptidase 4 (DPP4) inhibitors versus metformin. Our objective was to investigate whether the initiation of DPP4 inhibitors, as opposed to metformin, was linked to a reduced risk of dementia. METHODS: We included individuals with type 2 diabetes over 40 years old who were new users of DPP4 inhibitors or metformin in the Chinese Renal Disease Data System (CRDS) database between 2009 and 2020. The study employed Kaplan-Meier and Cox regression for survival analysis and the Fine and Gray model for the competing risk of death. RESULTS: Following a 1:1 propensity score matching, the analysis included 3626 DPP4 inhibitor new users and an equal number of metformin new users. After adjusting for potential confounders, the utilization of DPP4 inhibitors was associated with a decreased risk of all-cause dementia compared to metformin (hazard ratio (HR) 0.63, 95% confidence interval (CI) 0.45-0.89). Subgroup analysis revealed that the utilization of DPP4 inhibitors was associated with a reduced incidence of dementia in individuals who initiated drug therapy at the age of 60 years or older (HR 0.69, 95% CI 0.48-0.98), those without baseline macrovascular complications (HR 0.62, 95% CI 0.41-0.96), and those without baseline microvascular complications (HR 0.67, 95% CI 0.47-0.98). CONCLUSION: In this real-world study, we found that DPP4 inhibitors presented an association with a lower risk of dementia in individuals with type 2 diabetes than metformin, particularly in older people and those without diabetes-related comorbidities.

A case of immunoglobulin G4-related kidney disease manifesting after dipeptidyl peptidase-4 inhibitor treatment.

A 68-year-old man with type 2 diabetes mellitus was admitted with decreased renal function. He had high IgG4 (1070 mg/dL) and hypocomplementemia (CH50, 25 U/mL). Kidney biopsy showed tubulointerstitial nephritis with IgG4-positive plasma cell infiltration. Four years later, a second kidney biopsy revealed a new manifestation of membranous nephropathy and tubulointerstitial nephritis with exacerbated fibrosis formation. Six years later, the patient developed bullous pemphigoid, which was thought to be caused by DPP4 inhibitors, so DPP4 inhibitor treatment was discontinued. The use of DPP4 inhibitors correlated with changes in renal function, and the patient was diagnosed with IgG4-related kidney disease related to DPP4 inhibitors.

Efficacy and Safety of Vildagliptin for Type 2 Diabetes in Patients With Diabetic Kidney Disease.

BACKGROUND/AIM: Vildagliptin is one of the dipeptidyl peptidase-4 (DPP-4) inhibitors that have been shown to improve hyperglycemia in clinical trials among patients with type 2 diabetes. However, few studies have examined the efficacy of vildagliptin in patients with diabetic kidney disease (DKD). PATIENTS AND METHODS: Eight patients with DKD received oral vildagliptin 50-100 mg/day. The duration of diabetes was 6.7±5.9 years and observation period was 23.6±9.8 months. Changes in fasting blood glucose, and hemoglobin A1c (HbA1c), estimated glomerular filtration rate (eGFR), and urine protein-to-creatinine ratio (UPCR) were studied before and after the administration of vildagliptin. RESULTS: Vildagliptin treatment significantly decreased fasting blood glucose and HbA1c, compared to baseline (132±56 mg/dl, p=0.036, 6.0±0.3, p=0.041, respectively). UPCR tended to be decreased, albeit without statistical significance. However, eGFR was decreased after the administration of vildagliptin. No significant adverse effects were observed in all patients during the study. CONCLUSION: Although the sample size was limited and the observation period was brief, vildagliptin was found to be an effective and reasonably well-tolerated treatment for patients with DKD.

Association of sulfonylureas with the risk of dementia: A population-based cohort study.

BACKGROUND: Sulfonylureas are oral glucose-lowering medications positioned as a second-line therapy for type 2 diabetes. Evidence relating them to cognitive decline has been mixed. The objective was to determine whether sulfonylurea use was associated with a differential risk of dementia compared with dipeptidyl peptidase-4 (DPP4) inhibitor use. METHODS: Using administrative data from residents in Ontario, Canada, adults aged ≥66 years who were new users of a sulfonylurea or a DPP4 inhibitor from June 14, 2011, to March 31, 2021 entered this population-based retrospective cohort study. Dementia was ascertained using a validated algorithm for Alzheimer's disease and related dementias. Propensity-score weighted Cox proportional hazards models were used to obtain adjusted hazard ratios (aHR) and confidence intervals (CI) for time to incident dementia. The observation window started at 1 year after cohort entry to mitigate protopathic bias due to delayed diagnosis. The primary analysis used an intention-to-treat exposure definition. A separate propensity-score weighted analysis was conducted to explore within-class differences in dementia risk among sulfonylurea new users selected from the primary cohort. RESULTS: Among 107,806 DPP4 inhibitor new users and 37,030 sulfonylurea new users, sulfonylureas compared with DPP4 inhibitors were associated with a higher risk of dementia (18.4/1000 person-years; aHR [95% CI] = 1.09 [1.04-1.15]) over a mean follow-up of 4.82 years from cohort entry. Glyburide compared to gliclazide exhibited a higher dementia risk (aHR [95% CI] = 1.17 [1.03-1.32]). CONCLUSION: New use of a sulfonylurea especially glyburide was associated with a higher dementia risk compared with new use of a DPP4 inhibitor in older adults with diabetes.

Current findings on the efficacy of incretin-based drugs for diabetic kidney disease: A narrative review.

Diabetic kidney disease (DKD) is the most common cause of chronic kidney disease (CKD), leading end-stage renal disease. Thus, DKD is one of the most important diabetic complications. Incretin-based therapeutic agents, such as glucagon-like peptide-1 (GLP-1) receptor agonizts and dipeptidyl peptidase-4 (DPP-4) inhibitors have been reported to elicit vasotropic actions, suggesting a potential for effecting reduction in DKD. Glucose-dependent insulinotropic polypeptide (GIP) is also classified as an incretin. However, the insulin action after GIP secretion is known to be drastically reduced in patients with type 2 diabetes. Therefore, GIP has been formally considered unsuitable as a treatment for type 2 diabetes in the past. This concept is changing as it has been reported that resistance to GIP can be reversed and its effect restored with improved glycemic control. The development of novel dual- or triple- receptor agonizts that can bind to the receptors, not only for GLP-1 but also to GIP and glucagon receptors, is intended to simultaneously address several metabolic pathways including protein, lipid, and carbohydrate metabolism. These led to the development of GIP receptor agonist-based drugs for type 2 diabetes. The possibility of combined GIP/GLP-1 receptor agonist was also explored. The novel dual GIP and GLP-1 receptor agonist tirzepatide has recently been launched (Mounjaro®, Lilly). We have revealed precise mechanisms of the renoprotective effect of GLP-1 receptor agonizts or DPP-4 inhibitors, while the long-term effect of tirzepatide will need to be determined and its potential effects on kidneys should be properly tested.

Optimization of flaxseed milk fermentation for the production of functional peptides and estimation of their bioactivities.

Bioactive peptides are protein fragments which have a positive impact on the functions and conditions of living organisms. Apart from other animal and plant sources flaxseed is an excellent source of bioactive peptides. In recent years, fermentation has been explored as effective way for bioactive peptides generation. Hence, the present study has been carried out to evaluate an indigenous Lactobacillus plantarum strain NCDC 374 for fermentation and peptides generation in flaxseed milk. Optimization of fermentation condition to obtain maximum functional properties (Proteolytic activity, Antioxidant activity and ACE inhibition %) was investigated using response surface methodology. Optimal condition to produce the functional peptides were found to be 4.20% inoculum size with 126 hours of fermentation time. The fermented milk resulted in 67.38% inhibition in DPPH, 41.35% inhibition in ACE and 30.38 micro gram leucine/ml proteolytic activity. Molecular weight cut off membrane (Viva spin) were used to fractionate the peptides. 10 kDa peptides showed optimal results for % DPPH inhibition, ACE inhibition, Antimicrobial activity and DPP-IV inhibition as compared to 5 kDa.

Angioedema Caused by Drugs That Prevent the Degradation of Vasoactive Peptides: A Pharmacovigilance Database Study.

Angioedema results from the decreased degradation of vasoactive peptides such as substance P and bradykinin. In this study, we sought to clarify whether dipeptidyl peptidase-4 (DPP-4) and angiotensin-converting enzyme (ACE) inhibitors that suppress the degradation of substance P and bradykinin are involved in angioedema onset. We calculated information coefficients (ICs) by performing a disproportionality analysis to evaluate DPP-4/ACE inhibitor-induced angioedema using the Japanese Adverse Drug Event Report (JADER) database. No angioedema signals were detected for DPP-4 inhibitors; however, a signal was detected for ACE inhibitors (IC: 2.42, 95% confidence interval (CI): 2.19 to 2.65). Of the patients treated with DPP-4 inhibitors, four developed drug-induced angioedema in combination with ACE inhibitors, and all were taking vildagliptin. Signals were detected for enalapril (IC: 2.39, 95% CI: 2.06 to 2.71), imidapril (IC: 2.83, 95% CI: 2.38 to 3.27), lisinopril (IC: 2.28, 95% CI: 1.55 to 3.00), temocapril (IC: 1.35, 95% CI: 0.29 to 2.40), and trandolapril (IC: 1.57, 95% CI: 0.19 to 2.95). Both inhibitors inhibited the degradation of substance P and bradykinin and were thus expected to cause angioedema. However, no signal of angioedema was detected with the DPP-4 inhibitors, in contrast to some ACE inhibitors. This study found that ACE inhibitors and DPP-4 inhibitors, which inhibit the degradation of substance P and bradykinin, tended to have different effects on the onset of angioedema in clinical practice.

Efficacy and Cardiovascular Safety of DPP-4 Inhibitors.

Dipeptidyl peptidase-4 (DPP-4) inhibitors or gliptins belong to the class of incretin mimetics. These drugs have been available on the market for the management of type 2 diabetes mellitus (T2DM) for over a decade. Sitagliptin, linagliptin, vildagliptin, saxagliptin and alogliptin are widely available globally, whilst anagliptin, gemigliptin and teneliptin are used mainly in the Asian countries. The glycemic control conferred by DPP-4 inhibitors varies among individual molecules with an average reduction of glycated hemoglobin (HbA1c) ranging between -0.5 to -1.0% with monotherapy. Additive effects on HbA1c reduction may result from combination therapy with other antidiabetics. Weak evidence from various studies suggests that DPP-4 inhibitors may be useful in treating nonalcoholic fatty liver disease (NAFLD) and polycystic ovary syndrome (PCOS). DPP-4 inhibitors safety is not established in pregnancy, and there is only meagre evidence of its use in T2DM among children. In line with the United States Food and Drug Administration (US FDA) recommendations, sitagliptin, linagliptin, saxagliptin and alogliptin have undergone rigorous cardiovascular outcome trials (CVOTs) in recent years, and the safety data for vildagliptin is available through retrospective analysis of various studies in meta-analysis. Small clinical trial, and meta-analysis based data are available for the CV safety of other DPP-4 inhibitors. In general, the CVOTs and other safety data do not reveal serious warning signals except for saxagliptin (higher risk of hospitalization from heart failure [hHF]), although there is no robust data on the risk of hHF among patients with moderate to severe HF at baseline treated with other DPP-4 inhibitors. This review critically appraises the efficacy and cardiovascular safety of DPP-4 inhibitors to empower clinicians to use this class of antidiabetic medications judiciously.

The Effect of Glucagon-Like Peptide-1 Receptor Agonists on Renal Outcomes in Type 2 Diabetes.

This review examines the available literature on the effect of glucagon-like peptide-1 receptor agonists (GLP-1RAs) on renal outcomes in type 2 diabetes mellitus. Diabetes is an important cause of end-stage renal disease requiring renal replacement therapy, and diabetic kidney disease is an independent risk factor for cardiovascular disease (CVD). GLP-1RAs are proven to be safe in terms of CVD, and some of them have been shown to have a beneficial effect on cardiovascular outcomes. The effect of GLP-1RAs on hard renal endpoints has yet to be established; to date, there have been no published GLP-1RA clinical trials with primary renal endpoints. In this review, we discuss the evidence for a renal protective role of GLP-1RAs, highlighting the secondary renal outcomes from recent cardiovascular outcome trials of this class of glucose-lowering therapies.

Linagliptin safety profile: A systematic review.

OBJECTIVE: To describe the safety profile of linagliptin. METHODOLOGY: Systematic review using PubMed/MEDLINE, BVS and Web of Science. The search strategy "Linagliptin" AND "safety" was used. The inclusion criteria were clinical trials with a control group composed of conventional DM2 pharmacotherapy. RESULTS: We identified 16 studies, and the most frequent adverse events (AEs) were nasopharyngitis with linagliptin at 5 and 10mg in monotherapy (31.6% and 29.6%, respectively) and gastrointestinal events (>10.0%) with linagliptin in combination. Of the AEs, 14.9 (±3.1)% were associated with the use of linagliptin in monotherapy, and 17.6 (±6.0)% in combination. The linagliptin AEs have a varied occurrence and frequency, ranging from mild to moderate intensity.

The past decade in type 2 diabetes and future challenges.

There is today an exponential increase in prevalence of type 2 diabetes mellitus (T2DM), especially in young people. This downward shift in age of onset of T2DM has been shown by abundant evidence to be due to an increase in obesity among the young, the latter mainly attributable to unhealthy dietary habits and a sedentary lifestyle. It is therefore obvious that the prevention of diabetes rather its treatment is of is paramount importance. In the past decade, because concerns about the safety of antidiabetic agents took precedence over the issue of efficacy, almost all studies have been diabetes CVOTs and not traditional CVOTs. Until 2015, the evidence showed that antidiabetic agents are effective in terms of reduction of microvascular, as opposed to macrovascular, complications. However, following publication of the results of some new studies, it became clear that the new class of antidiabetic drugs, e.g., SGLT 2 inhibitors and GLP-1 agonists, are also effective in reducing cardiovascular disease (CVD). In the coming decade, numerous health challenges are expected to arise, the most important being the greater expansion of the therapeutic armamentarium for T2DM and the adoption of strategies for prevention of CVDs. In parallel, the new generation of antidiabetic agents will target the recently investigated pathophysiologic disorders of diabetes, while, ideally, treatments should include smart drugs without side effects.

Is there a Chance to Promote Arteriogenesis by DPP4 Inhibitors Even in Type 2 Diabetes? A Critical Review.

Cardiovascular diseases (CVD) are still the prevailing cause of death not only in industrialized countries, but even worldwide. Type 2 diabetes mellitus (type 2 DM) and hyperlipidemia, a metabolic disorder that is often associated with diabetes, are major risk factors for developing CVD. Recently, clinical trials proved the safety of gliptins in treating patients with type 2 DM. Gliptins are dipeptidyl-peptidase 4 (DPP4/CD26) inhibitors, which stabilize glucagon-like peptide-1 (GLP-1), thereby increasing the bioavailability of insulin. Moreover, blocking DPP4 results in increased levels of stromal cell derived factor 1 (SDF-1). SDF-1 has been shown in pre-clinical animal studies to improve heart function and survival after myocardial infarction, and to promote arteriogenesis, the growth of natural bypasses, compensating for the function of an occluded artery. Clinical trials, however, failed to demonstrate a superiority of gliptins compared to placebo treated type 2 DM patients in terms of cardiovascular (CV) outcomes. This review highlights the function of DPP4 inhibitors in type 2 DM, and in treating cardiovascular diseases, with special emphasis on arteriogenesis. It critically addresses the potency of currently available gliptins and gives rise to hope by pointing out the most relevant questions that need to be resolved.

Pharmacological Treatment in Diabetes Mellitus Type 1 - Insulin and What Else?

The basis of treatment in autoimmune diabetes is insulin therapy; however, many clinical cases have proven that this method does not solve all problems. Trials of causal treatment including blocking the autoimmune processes and insulin-producing cells transplants were carried out. Those methods require more research to be concerned as efficient and safe ways of treatment in type 1 diabetes. The use of non-insulin adjunct treatment is a new trend. It has been successfully used in laboratories as well as clinical trials. Metformin is the most widely used drug, together with sodium-glucose co-transporters 2 (SGLT2) inhibitors, amylin analogues, glucagon-like peptide 1 (GLP-1) receptor agonists, and dipeptidyl peptidase-4 (DPP-4) inhibitors. The results of administration of these medicaments give good outcomes in patients with diabetes mellitus type 1. Most likely, in the near future, they will progressively be used in both adult and adolescent patients with type 1 diabetes. Further multicenter, randomized studies are required to evaluate the efficacy of treatment and long term safety of these drugs.

Clinical Safety and Tolerability of Vildagliptin - Insights from Randomised Trials, Observational Studies and Post-marketing Surveillance.

Vildagliptin is one of the most extensively studied dipeptidyl peptidase-4 (DPP-4) inhibitors in terms of its clinical utility. Over the last decade, a vast panorama of evidence on the benefit-risk profile of vildagliptin has been generated in patients with type 2 diabetes mellitus (T2DM). In this article, we review the cumulative evidence on the safety of vildagliptin from the clinical development programme, as well as reports of rare adverse drug reactions detected during the post-marketing surveillance of the drug. Across clinical studies, the overall safety and tolerability profile of vildagliptin was similar to placebo, and it was supported by real-world data in a broad population of patients with T2DM, making DPP-4 inhibitors, like vildagliptin, a safe option for managing patients with T2DM.

The Vildagliptin Experience - 25 Years Since the Initiation of the Novartis Glucagon-like Peptide-1 Based Therapy Programme and 10 Years Since the First Vildagliptin Registration.

The discovery of the incretin hormone glucagon like peptide-1 (GLP-1), and its usefulness in the treatment of type 2 diabetes mellitus (T2DM) followed by the finding that dipeptidyl peptidase-4 (DPP-4) inhibition prevents GLP-1 inactivation, led to the discovery of DPP-728. In 1999, studies with DPP-728 established the first proof-of-concept that DPP-4 inhibition improves glycaemic control in patients with T2DM. Further efforts to improve the binding kinetics of DPP-728 resulted in the discovery of vildagliptin (LAF237). In the last 20 years, a plethora of studies conducted by Novartis in collaboration with external investigators has demonstrated the mechanism of action of vildagliptin and its efficacy as monotherapy and as an add-on therapy for patients with T2DM. The studies establish that vildagliptin is a selective DPP-4 inhibitor that blocks GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) inactivation, thereby prolonging their action, resulting in improved glycaemic control. This review aims to discuss the discovery and development of vildagliptin, with an emphasis on mechanism of action and clinical efficacy.