An update of SGLT1 and SGLT2 inhibitors in early phase diabetes-type 2 clinical trials

Introduction: More than 424 million adults have diabetes mellitus (DM). This number is expected to increase to 626 million by 2045. The majority (90–95%) of people with DM has type 2-diabetes (T2DM). The continued prevalence of DM and associated complications has prompted investigators to find new therapies. One of the most recent additions to the anti-diabetic armamentarium are inhibitors of sodium-glucose co-transporters 1 and 2 (SGLT1, SGLT2).

Areas covered: The authors review the status of SGLT2 inhibitors for the treatment of T2DM and place an emphasis on those agents in early phase clinical trials. Data and information were retrieved from American Diabetes Association, Diabetes UK, ClinicalTrials.gov, PubMed, and Scopus websites. The keywords used in the search were T2DM, SGLT1, SGLT2, and clinical trials.

Expert opinion: The benefits of SGLT inhibitors include reductions in serum glycated hemoglobin (HbA1c), body weight, blood pressure and cardiovascular and renal events. However, SGLT inhibitors increase the risk of genitourinary tract infections, diabetic ketoacidosis, and bone fractures. The development of SGLT inhibitors with fewer side effects and as combination therapies are the key to maximizing the therapeutic effects of this important class of anti-diabetic drug.     

Bone Mass and Microarchitecture in CKD Patients with Fracture

Patients with predialysis chronic kidney disease (CKD) have increased risk for fracture, but the structural mechanisms underlying this increased skeletal fragility are unknown. We measured areal bone mineral density (aBMD) by dual-energy x-ray absorptiometry at the spine, hip, and radius, and we measured volumetric BMD (vBMD), geometry, and microarchitecture by high-resolution peripheral quantitative computed tomography (HR-pQCT) at the radius and tibia in patients with CKD: 32 with fracture and 59 without fracture. Patients with fracture had lower aBMD at the spine, total hip, femoral neck, and the ultradistal radius, the last having the strongest association with fracture. By HR-pQCT of the radius, patients with fracture had lower cortical area and thickness, total and trabecular vBMD, and trabecular number and greater trabecular separation and network heterogeneity. At the tibia, patients with fracture had significantly lower cortical area, thickness, and total and cortical density. Total vBMD at both radius and tibia most strongly associated with fracture. By receiver operator characteristic curve analysis, patients with longer duration of CKD had area under the curve of >0.75 for aBMD at both hip sites and the ultradistal radius, vBMD and geometry at the radius and tibia, and microarchitecture at the tibia. In summary, patients with predialysis CKD and fractures have lower aBMD by dual-energy x-ray absorptiometry and lower vBMD, thinner cortices, and trabecular loss by HR-pQCT. These density and structural differences may underlie the increased susceptibility to fracture among patients with CKD.Fracture rates in patients with ESRD are elevated,¹ as high as individuals who have normal kidney function and are older by 10 to 20 years.² Recently, there has been increasing recognition that patients with predialysis chronic kidney disease (CKD) also experience an increased fracture burden.^2–5 In 2006, we reported that participants who were older than 50 years in the Third National Health and Nutrition Examination Survey (NHANES III) and had an estimated GFR (eGFR) between 15 and 59 ml/min (stages 3 and 4 CKD) had a two-fold higher risk for hip fracture than individuals without CKD.⁶ Subsequent studies confirmed our findings and also demonstrated that fracture risk increases as kidney function declines.^3–5 In one study, hip fracture risk was as high in patients with stage 4 CKD as in patients with ESRD.⁴ Given the rapid expansion of the population of individuals who are older than 65 years worldwide and the high prevalence of CKD in the elderly,⁷ it is highly important to improve our understanding of the structural and biologic mechanisms that contribute to increased fracture rates in patients with CKD so that we can develop strategies to identify those who are at risk for fracture.In patients with ESRD, relationships between areal bone mineral density (aBMD) measured by dual-energy x-ray absorptiometry (DXA) and prevalent fractures are inconsistent; some studies detected no difference in aBMD,^8–11 whereas others, including a meta-analysis, found lower aBMD in those with prevalent spine and nonspine fractures^8,11,12; therefore, recent Kidney Disease: Improving Global Outcomes (KDIGO) guidelines for the diagnosis and management of CKD-mineral and bone disorder (CKD-MBD) do not recommend routine measurement of aBMD by DXA in patients with ESRD or in those with late stages 3 through 5 CKD, because more severe CKD is commonly associated with renal osteodystrophy.¹³ In contrast, however, KDIGO guidelines recommend DXA to assess fracture risk in patients with stage 1 through early stage 3 CKD, as long as biochemical testing does not suggest CKD-MBD.Fractures occur when bone strength is not sufficient to withstand an applied force. Bone strength is determined both by the density of bone present and by the quality of that bone. Whereas bone density is measured by DXA, other factors that contribute to bone quality, such as cortical and trabecular microarchitecture, are not. Chronic parathyroid hormone excess, a common biochemical feature of CKD, is generally associated with trabecularization of endocortical bone, cortical thinning, and increased cortical porosity.^14–16 Whereas effects of hyperparathyroidism on trabecular bone are less consistent, trabecular structure and connectivity are generally maintained.¹⁴ The resolution of DXA is too low to distinguish between cortical and trabecular bone, both of which influence resistance to fracture; this may account for its inconsistent utility in discriminating fracture status in patients with ESRD.Bone biopsy studies have demonstrated that histomorphometric abnormalities of renal osteodystrophy may begin early in the development of CKD¹⁷; therefore, DXA may also have limited utility in patients with predialysis kidney disease. Recently, Bacchetta et al.¹⁸ used high-resolution peripheral quantitative computed tomography (HR-pQCT; voxel size 82 μm) to demonstrate that both cortical and trabecular microarchitecture are abnormal in patients with early CKD compared with healthy control subjects. In addition, they reported thinner cortices and abnormal trabecular microarchitecture in a small number of patients with CKD and with fractures; however, they did not measure aBMD by DXA. In this study, we measured aBMD by DXA and volumetric BMD (vBMD) and bone microarchitecture by HR-pQCT in patients with predialysis CKD (stages 2 through 5), with and without prevalent fracture. On the basis of results in postmenopausal women with normal kidney function reported by Boutroy et al.¹⁹ and by our group,²⁰ we hypothesized that HR-pQCT would detect abnormalities of vBMD and bone geometry and microstructure in patients with CKD and a history of fracture, whereas measurement of aBMD by DXA would not.     

Individual trabeculae segmentation (ITS)–based morphological analysis of high‐resolution peripheral quantitative computed tomography images detects abnormal trabecular plate and rod microarchitecture in premenopausal women with idiopathic osteoporosis

Idiopathic osteoporosis (IOP) in premenopausal women is a poorly understood entity in which otherwise healthy women have low‐trauma fracture or very low bone mineral density (BMD). In this study, we applied individual trabeculae segmentation (ITS)–based morphological analysis to high‐resolution peripheral quantitative computed tomography (HR‐pQCT) images of the distal radius and distal tibia to gain greater insight into skeletal microarchitecture in premenopausal women with IOP. HR‐pQCT scans were performed for 26 normal control individuals and 31 women with IOP. A cubic subvolume was extracted from the trabecular bone compartment and subjected to ITS‐based analysis. Three Young's moduli and three shear moduli were calculated by micro–finite element (µFE) analysis. ITS‐based morphological analysis of HR‐pQCT images detected significantly decreased trabecular plate and rod bone volume fraction and number, decreased axial bone volume fraction in the longitudinal axis, increased rod length, and decreased rod‐to‐rod, plate‐to‐rod, and plate‐to‐plate junction densities at the distal radius and distal tibia in women with IOP. However, trabecular plate and rod thickness did not differ. A more rod‐like trabecular microstructure was found in the distal radius, but not in the distal tibia. Most ITS measurements contributed significantly to the elastic moduli of trabecular bone independent of bone volume fraction (BV/TV). At a fixed BV/TV, plate‐like trabeculae contributed positively to the mechanical properties of trabecular bone. The results suggest that ITS‐based morphological analysis of HR‐pQCT images is a sensitive and promising clinical tool for the investigation of trabecular bone microstructure in human studies of osteoporosis. © 2010 American Society for Bone and Mineral Research     

Methods for detecting DNA methylation in tumors: from bench to bedside

Tumor-acquired changes in DNA methylation are the focus of research in an increasing number of basic, translational, and clinical laboratories around the world. In the laboratory, genome-wide technologies such as expression and DNA microarrays have been adapted to analyze patterns of DNA methylation and to screen for novel disease markers. Other technologies that are relatively inexpensive and highly sensitive such as methylation-specific PCR (MSP), or quantitative, such as quantitative MSP and pyrosequencing are widely used in retrospective studies and have potential in a diagnostic setting. In the near future, it may be possible to screen patients for common cancers using DNA methylation signatures as well as to measure patient responses to treatment, to identify patients at increased risk, or to monitor interventions designed to reduce cancer incidence. In this article, we review genome-wide and quantitative, high- resolution methods for methylation analysis that are used in the laboratory and clinic, and discuss their potential for use in a clinical setting.     

Methylation and Gene Silencing of the Ras-Related GTPase Gene in Lung and Breast Cancers

Background RRAD, a small Ras-related GTPase, is highly expressed in human skeletal muscle, lung, and heart. Although loss of expression of RRAD in breast cancer cells has been reported and it may act as an oncogene, the mechanism of silencing is unknown. Methods We examined (1) mRNA expression of RRAD in lung and breast cancer cell lines using RT-PCR and (2) methylation status of lung and breast cancers. Results Loss of RRAD expression was found in 14 of 20 (70%) NSCLC cell lines, 11 of 11 (100%) SCLC cell lines, and 8 of 10 (80%) breast cancer cell lines; expression was not affected in normal bronchial and mammary epithelial cells. Treatment of 23 expression-negative cell lines with a demethylating agent restored expression in all cases. We developed a methylation-specific assay from the analysis of bisulfite sequencing of the 5′ region of RRAD in expression-negative and positive cell lines, which resulted in good concordance between methylation and expression. Primary lung and breast cancers showed hypermethylation in 89 of 214 (42%) and 39 of 63 (62%) cases, respectively. RRAD hypermethylation correlated with smoking history and poorer prognosis in lung adenocarcinomas. Conclusions We conclude that epigenetic silencing of RRAD is a frequent event in lung and breast cancers, and analysis of it may provide novel opportunities for prognosis and therapy of these cancers.     

Evidence for alternative candidate genes near RB1 involved in clonal expansion of in situ urothelial neoplasia

In this paper, we present whole-organ histologic and genetic mapping studies using hypervariable DNA markers on chromosome 13 and then integrate the recombination- and single-nucleotide polymorphic sites (SNPs)-based deletion maps with the annotated genome sequence. Using bladders resected from patients with invasive urothelial carcinoma, we studied allelic patterns of 40 microsatellite markers mapping to all regions of chromosome 13 and 79 SNPs located within the 13q14 region containing the RB1 gene. A whole-organ histologic and genetic mapping strategy was used to identify the evolution of allelic losses on chromosome 13 during the progression of bladder neoplasia. Markers mapping to chromosomal regions involved in clonal expansion of preneoplastic intraurothelial lesions were subsequently tested in 25 tumors and 21 voided urine samples of patients with bladder cancer. Four clusters of allelic losses mapping to distinct regions of chromosome 13 were identified. Markers mapping to the 13q14 region that is flanked by D13S263 and D13S276, which contains the RB1 gene, showed allelic losses associated with early clonal expansion of intraurothelial neoplasia. Such losses could be identified in approximately 32% bladder tumor tissue samples and 38% of voided urines from patients with bladder cancer. The integration of distribution patterns of clonal allelic losses revealed by the microsatellite markers with those obtained by genotyping of SNPs disclosed that the loss within an approximately 4-Mb segment centered around RB1 may represent an incipient event in bladder neoplasia. However, the inactivation of RB1 occurred later and was associated with the onset of severe dysplasia/carcinoma in situ. Our studies provide evidence for the presence of critical alternative candidate genes mapping to the 13q14 region that are involved in clonal expansion of neoplasia within the bladder antecedent to the inactivation of the RB1 gene.     

Gain of a region on 7p22.3, containing MAD1L1, is the most frequent event in small-cell lung cancer cell lines

Small-cell lung cancer (SCLC) is a highly aggressive lung neoplasm, which accounts for 20% of yearly lung cancer cases. The lack of knowledge of the progenitor cell type for SCLC precludes the definition of a normal gene expression profile and has hampered the identification of gene expression changes, while the low resolution of conventional genomic screens such as comparative genomic hybridization (CGH) and loss of heterozygosity analysis limit our ability to fine-map genetic alterations. The recent advent of whole genome tiling path array CGH enables profiling of segmental DNA copy number gains and losses at a resolution 100 times that of conventional methods. Here we report the analysis of 14 SCLC cell lines and six matched normal B-lymphocyte lines. We detected 7p22.3 copy number gain in 13 of the 14 SCLC lines and 0 of the 6 matched normal lines. In 4 of the 14 cell lines, this gain is present as a 350 kbp gene specific copy number gain centered at MAD1L1 (the human homologue of the yeast gene MAD1). Fluorescence in situ hybridization validated the array CGH finding. Intriguingly, MAD1L1 has been implicated to have tumor-suppressing functions. Our data suggest a more complex role for this gene, as MAD1L1 is the most frequent copy number gain in SCLC cell lines.     

Met Activation in Non-Small Cell Lung Cancer Is Associated with de Novo Resistance to EGFR Inhibitors and the Development of Brain Metastasis

Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors.Activating mutations in EGFR (primarily del19 EGFR and L858R) are associated with sensitivity to epidermal growth factor receptor (EGFR) small molecule tyrosine kinase inhibitor therapy, but patients uniformly develop disease recurrence.^1,2,3,4,5,6 In addition, about 30% of patients with EGFR sensitizing mutations show primary resistance to EGFR inhibitor therapy.^{1,2,3,4,5,6,7} While disease recurrence in formerly responsive patients has been associated with an EGFRT790M mutation,⁸ MET amplification,^9,10 or hepatocyte growth factor (HGF) expression,¹¹ the factors involved in de novo resistance remain unidentified. Sequist et al reported primary resistance to EGFR tyrosine kinase inhibitor (TKI) in a patient harboring Met copy gain, but analysis of larger cohorts has not been reported and Met protein expression and activation were not tested.¹² We examined a cohort of EGFR mutant non-small cell lung cancer (NSCLC) patients before treatment with EGFR TKI (designated cohort 1), and we report here that Met protein expression and phosphorylation were found in a subset of tumors before EGFR TKI therapy. Importantly, the subset harboring Met expression and phosphorylation was associated with poor response to subsequent EGFR TKI therapy despite the presence of EGFR inhibitor sensitizing mutations.In addition to primary resistance, poor outcome to EGFR targeted therapy in NSCLC can result from development of metastases, especially to the central nervous system. Since HGF/Met signaling is uniquely positioned to be a key factor in cell migration and tumor dissemination,^13,14 we compared Met status in a separate cohort of NSCLC patients (designated cohort 2) with paired brain metastases. Met expression has been correlated with both development of metastases and poor prognosis in some tumor types,¹³ but no studies have demonstrated Met activation in metastatic lung cancer. In this study we found that Met expression and phosphorylation in primary NSCLC tumors were strongly associated with subsequent development of brain metastases. Furthermore, we showed an enrichment of cells positive for Met expression and phosphorylation in brain lesions compared with matched primary lung tumors.