A Predominant Oxidative Renal Metabolite of Empagliflozin in Male Mice Is Cytotoxic in Mouse Renal Tubular Cells but not Genotoxic

Abstract

In a previously reported CD-1 mouse 2-year carcinogenicity study with the sodium glucose cotransporter-2 inhibitor empagliflozin, an increased incidence of renal tubular adenomas and carcinomas was identified only in the male high-dose group. Follow-up investigative studies have shown that the renal tumors in male high-dose mice were preceded by a number of renal degenerative/regenerative findings. Prior cross-species in vitro metabolism studies using microsomes identified an oxidative metabolite (M466/2) predominantly formed in the male mouse kidney and which spontaneously degrades to a metabolite (M380/1) and reactive 4-OH crotonaldehyde (CTA). In order to further evaluate potential modes of action for empagliflozin-associated male mouse renal tumors, we report here a series of in vitro investigative toxicology studies conducted to evaluate the cytotoxic and genotoxic potential of empagliflozin and M466/2. To assess the cytotoxic potential of empagliflozin and M466/2, a primary mouse renal tubular epithelial (mRTE) cell model was used. In mRTE cells, M466/2-derived in vitro 4-OH CTA exposure was cytotoxic, while empagliflozin was not cytotoxic or mitogenic. Empagliflozin and M466/2 were not genotoxic, supporting an indirect mode of action for empagliflozin-associated male mouse renal tumorigenesis. In conclusion, these in vitro data show that M466/2-derived 4-OH CTA exposure is associated with cytotoxicity in renal tubule cells and may be involved in promoting compound-related in vivo renal metabolic stress and chronic low-level renal injury, in turn supporting a nongenotoxic mode of tumor pathogenesis specific to the male mouse.

No Cytotoxic and Inflammatory Effects of Empagliflozin and Dapagliflozin on Primary Renal Proximal Tubular Epithelial Cells under Diabetic Conditions In Vitro

“…Several in vitro studies using immortalized human tubular epithelial cells (e.g., HK2 cell line) [25][26][27][28], murine tubular epithelial cells [29] or non-renal cells [30,31] investigated either the effects of empa-or of dapagliflozin. Some of them have shown that SGLT2 inhibition reduced the release of inflammatory or fibrotic factors induced by high glucose levels [25][26][27], others found effects on oxidative stress responses [26,30,31].…”

Dysplastic urothelial changes accompany empagliflozin administration in urinary bladder of experimental diabetes

“…Pathological changes started by apoptosis and increased mitotic figures then progressed to tubular hyperplasia, karyomegaly and increased Ki‐67 expression without abnormal kidney function . These changes might be attributed to EMPA metabolite, (M466/2)‐derived 4‐OH crotonaldehyde (CTA), which promotes oxidative stress as a mechanism for carcinogenesis . This is unlikely to occur in humans as glucuronidation is the main pathway for EMPA metabolism …”

“…8,29 These changes might be attributed to EMPA metabolite, (M466/2)-derived 4-OH crotonaldehyde (CTA), which promotes oxidative stress as a mechanism for carcinogenesis. 7 This is unlikely to occur in humans as glucuronidation is the main pathway for EMPA metabolism. 49 Besides oxidative stress, EMPA can induce changes in genes and p53-dependent pathways of cell cycle and proliferation.…”

“…6 Another important safety concern is the increased incidence of renal and testicular tumours associated with highdose EMPA in male mice. [7][8][9] Although a study by Tang et al 10 showed no significant association between SGLT2is and overall increased cancer risk in humans, EMPA is associated with increased risk of urinary bladder cancer (UC). 10 Additionally, the compelling evidence of DM association with UC [11][12][13][14] adds a further complexity to UC risk interpretation.…”

Mechanisms of Rodent Renal Carcinogenesis Revisited

“…M466/2 could not be identified in human kidney microsomes and was minimally present in rat microsomes (Taub et al 2015). M466/2 spontaneously degraded under physiologic conditions to a reactive species, 4-hydroxycrotonaldehyde (4-OH CTA), and a phenol metabolite (Smith et al 2017). 4-OH CTA has been shown to be cytotoxic for renal tubule epithelial cells, but is not genotoxic (Smith et al 2017).…”