Carcinogenicity in rats of the SGLT2 inhibitor canagliflozin

doi:10.1016/j.cbi.2014.09.018

Chemico-Biological Interactions

Volume 224, 5 December 2014, Pages 1-12

https://doi.org/10.1016/j.cbi.2014.09.018 Get rights and content

Highlights

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Canagliflozin, an SGLT2 inhibitor for treatment of diabetes, caused tumors in rats.
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Leydig cell tumors were attributed to increased luteinizing hormone.
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Adrenal and renal tumors were attributed to carbohydrate malabsorption and alterations in calcium homeostasis.

Abstract

The carcinogenicity potential of canagliflozin, an inhibitor of SGLT2, was evaluated in a 2-year rat study (10, 30, and 100 mg/kg). Rats showed an increase in pheochromocytomas, renal tubular tumors, and testicular Leydig cell tumors. Systemic exposure multiples at the highest dose relative to the maximum clinical dose were 12- to 21-fold. Pheochromocytomas and renal tubular tumors were noted in both sexes at 100 mg/kg. Leydig cell tumors were observed in males in all dose groups and were associated with increased luteinizing hormone levels. Hyperplasia was increased in the adrenal medulla at 100 mg/kg, but only a limited increase in simple tubular hyperplasia was observed in the kidney of males at 100 mg/kg. Hyperostosis occurred and was accompanied by substantial effects on calcium metabolism, including increased urinary calcium excretion and decreased levels of calcium regulating hormones (1,25-dihydroxyvitamin D and parathyroid hormone). A separate study with radiolabeled calcium confirmed that increased urinary calcium excretion was mediated via increased calcium absorption from the gastrointestinal tract. It was hypothesized that, at high doses, canagliflozin might have inhibited glucose absorption in the intestine via SGLT1 inhibition that resulted in glucose malabsorption, which increased calcium absorption by stimulating colonic glucose fermentation and reducing intestinal pH. Pheochromocytomas and adrenal medullary hyperplasia were attributed to altered calcium homeostasis, which have a known relationship in the rat. In conclusion, Leydig cell tumors were associated with increased luteinizing hormone levels and pheochromocytomas were most likely related to glucose malabsorption and altered calcium homeostasis. Renal tubular tumors may also have been linked to glucose malabsorption.

Introduction

SGLT2, a high-capacity, low-affinity glucose transporter, is expressed primarily in the proximal renal tubule and is responsible for most glucose reabsorption in the kidneys [1]. The transport of glucose is against its concentration gradient, coupled to the active transport of sodium. Inhibition of SGLT2 decreases glucose reabsorption in the renal tubule, increases urinary glucose excretion, and thereby reduces plasma glucose [1]. Phlorizin, which is a nonselective inhibitor of SGLT1 (another isoform which is predominantly present in the gastrointestinal tract) and SGLT2, reduces plasma glucose in preclinical models of type 2 diabetes mellitus (T2DM) [2]. Due to its nonselective nature, with activity on SGLT1, SGLT2, and glucose transporters and its poor pharmaceutical properties, phlorizin is unsuitable for clinical development [3]. A number of selective, metabolically stable, SGLT2 inhibitors have been discovered and are in clinical development to treat T2DM [4]. Canagliflozin was the first SGLT2 inhibitor approved in the United States for the treatment of T2DM.

Canagliflozin is a potent inhibitor of SGLT2 activity and has low potency SGLT1 inhibitory activity. Canagliflozin has been shown to increase urinary glucose excretion and decrease plasma glucose in animal models and in humans with T2DM [5], [6]. In repeated-dose toxicity studies with canagliflozin in mice up to 3 months, rats up to 6 months, and dogs up to 12 months, the compound was well tolerated and preneoplastic lesions were not observed [7], [8]. In these studies, there were substantial effects on urinalysis parameters that were considered to be related to the mode of action. These effects included increases in urinary volume, glucose, and electrolytes. Rats appeared to be the most sensitive species, with additional effects on calcium homeostasis and bone that were not observed in mice or dogs.

This report describes the preclinical assessment of the carcinogenicity potential of canagliflozin that was conducted as part of the development program to support world-wide registration. Where applicable, the results of the 3-month toxicity study in rats will be used for interpretation of findings in the carcinogenicity study. To assist interpretation of marked changes in calcium homeostasis (i.e., increased urinary excretion of calcium and increased trabecular bone formation) in rats treated with canagliflozin, a radiolabeled calcium absorption study was conducted in rats and the results are discussed in this article. In addition, possible mechanisms for induction of tumors in rats by canagliflozin and its clinical relevance are discussed in this article. Additional investigative studies that support the proposed mechanism of tumor formation are reported in a companion paper [9].

Section snippets

Test substance

Canagliflozin (CAS# 842133-18-0) with assay purity of 98.0% was formulated as an aqueous suspension containing 0.5% w/v hydroxypropyl methylcellulose in water. Formulations were stored refrigerated (2–8 °C) and in the dark. The concentrations, homogeneity, and stability of the formulations were checked during the studies using liquid chromatography and were confirmed to be within acceptance criteria.

Animals

Studies were performed with Sprague-Dawley rats purchased from Charles River (Kingston, NY and

Body weight, food consumption, and clinical observations

Dose-related decreases were observed in body weight and body weight gain throughout the study. Decreases were especially pronounced in the high-dose group, with terminal body weights and body weight gains of 0.79× and 0.70× for males and 0.77× and 0.67× for females, respectively, compared to vehicle control rats that received 0.5% w/v hydroxypropyl methylcellulose without canagliflozin. Food consumption was increased in a generally dose-related manner during the entire study. In males/females at

Discussion

The carcinogenicity profile of canagliflozin showed treatment-related tumors in three target organs in rats. These consisted of RTTs, pheochromocytomas, and LCTs. Canagliflozin is not genotoxic based on a battery of in vitro (Ames, mouse lymphoma) and in vivo (micronucleus, Comet) tests [7], [8]. Therefore, the tumors are considered to occur by a non-genetic mechanism.

Basophilic RTTs (adenomas and carcinomas) were noted in both sexes at 100 mg/kg. The 2 RTTs (1 benign, 1 malignant) in males at 30

Conclusion

In conclusion, the carcinogenicity potential of canagliflozin was investigated in a 2-year study in rats. Three types of treatment-related tumors (LCTs, pheochromocytomas, and RTTs) were observed. LCTs were attributed to increased LH, an established mechanism in rats. Pheochromocytomas were attributed primarily to altered calcium homeostasis following increased calcium absorption secondary to carbohydrate malabsorption. RTTs may also have been linked to carbohydrate malabsorption, and

Funding

This work was supported by Janssen Research & Development, LLC and Janssen Research & Development, a Division of Janssen Pharmaceutica NV. The authors are all employees and, as such, designed the studies, participated in data collection and analysis, made the decision to publish, and prepared the manuscript.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

Acknowledgements

The authors are grateful to Dr. Richard Sharpe (University of Edinburgh) for advice on the mechanistic studies related to LCTs and Dr. Terry Nett (Colorado State University) for assistance with the mechanistic studies and for the conduct of assays for testosterone and LH. The authors are grateful to Dr. Gordon Hard and Dr. Jerry Hardisty (Experimental Pathology Laboratories) for assistance with microscopic evaluation of the kidneys. The authors thank Verna Hillsamer (Janssen Research &

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The CREDENCE trial testing canagliflozin and the EMPA-REG OUTCOME trial testing empagliflozin suggest different effects on acute kidney injury (AKI). AKI diagnosis was mainly made based on changes of serum creatinine (sCr) although this also reflect mode of action of SGLT-2 inhibitors. We analyzed both compounds in a rat AKI model. The renal ischemia-reperfusion injury (I/R) model was used. Four groups were analyzed: sham, I/R+placebo, I/R+canagliflozin (30 mg/kg/day), I/R+ empagliflozin (10 mg/kg/day). Glucose excretion was comparable in both treatment groups indicating comparable SGLT-2 inhibition. Comparing GFR surrogate markers after I/R (sCr and blood urea nitrogen (BUN)), sCr peaked 24 h after I/R, BUN after 48 h, respectively, in the placebo treated I/R group. At all investigated time points after I/R sCr and BUN was higher in the I/R + canagliflozin group as compared to placebo treated rats, whereas the empagliflozin group did not differ from the placebo group. I/R led to tubular dilatation and necrosis. Empagliflozin was able to reduce that finding whereas canagliflozin had no effect. Treatment with empagliflozin also resulted in a significant reduction in an improved inflammatory score (p = 0.006). Renal expression of kidney injury molecule-1 (KIM-1) increased after I/R and empagliflozin but not canagliflozin significantly alleviated KIM-1 expression. I/R reduced urinary miR-26a excretion. Empagliflozin but not canagliflozin was able to restore normal levels of urinary miR-26a. This study in an AKI model confirmed safety data in the EMPA-REG OUTCOME trial suggesting that empagliflozin might reduce AKI risk. The empagliflozin effects on KIM-1 and miR-26a might indicate beneficial regulation of inflammation. These data should stimulate clinical studies with AKI risk as primary endpoint.
Effectiveness in the inhibition of dapagliflozin and canagliflozin on M-type K<sup>+</sup> current and α-methylglucoside-induced current in pituitary tumor (GH<inf>3</inf>) and pheochromocytoma PC12 cells
2020, European Journal of Pharmacology
Citation Excerpt :
One may speculate that the IK(M) detected in GH3 cells be distinguishable from that existing in different types of endocrine or neuroendocrine cells. An earlier report showed that the administration of CANA, inhibitor of SGLT2, could increase the incidence of pheochromocytomas in rats (De Jonghe et al., 2014). The possible effect of DAPA on IK(M) in pheochromocytoma PC12 cells were thus further investigated.
Dapagliflozin (DAPA) or canagliflozin (CANA), Na⁺-dependent glucose co-transporter type 2 (SGLT2) inhibitors, were used for treatment of type II diabetes mellitus. Addition of DAPA or CANA suppressed M-type K⁺ current (I_K(M)) in pituitary tumor (GH₃) and pheochromocytoma PC12 cells. The IC₅₀ value for DAPA- or CANA-mediated inhibition of I_K(M) in GH₃ cells was 0.11 or 0.42 μM, respectively. The presence of DAPA (0.1 μM) shifted the steady-state activation of I_K(M) to less depolarized potential without changing the gating charge of the current. During high-frequency depolarizing pulses, I_K(M) magnitude was reduced by DAPA; however, DAPA-induced block of I_K(M) remained effective. The amplitude of neither erg-mediated K⁺ current nor hyperpolarization-activated cation current in GH₃ cells was modified in the presence of 1 μM DAPA. Alternatively, addition of DAPA, CANA, phlorizin or chlorotoxin effectively suppressed α-methylglucoside-(αMG-) induced current (I_αMG) in GH₃ cells, albeit inability of tefluthrin (activator of I_Na) to suppress this current. DAPA shifted the charge-voltage relation of presteady-state I_αMG in a rightward and downward direction with no change in the gating charge of the I_αMG. Under current-clamp recordings, subsequent additions of DAPA, but still in the continued presence of αMG, increased the firing rate of spontaneous action potentials stimulated by αMG. Our results suggested that activity of SGLT was expressed functionally in GH₃ and PC12 cells. Therefore, inhibitory actions of DAPA or CANA on the amplitude and gating of I_K(M) might provide a yet unidentified mechanism through which the SGLT1 or SGLT2 activity were attenuated in unclamped cells occurring in vivo.
Promise, peril, and possible new treatment options incurred by sodium glucose co-transporter 2 (SGLT2)inhibitors: A precise review up to 2018
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The sodium-glucose co-transporter 2 inhibitors (SGLT2 inhibitors) are newly introduced adjuvant antidiabetic medications that block the reabsorption of glucose in the area of proximal tubule of the kidney. In this review, we pointed out all the beneficial (promises) and unwanted (perils) effects of SGLT2 inhibitors recorded in the groups with diabetes mellitus and animal models for developing risk benefit idea. After reviewing sufficient literature we also put forward the diseases those can be the novel indications of SGLT2 inhibitors. Review also revealed that besides Type 2 diabetes (T2D), SGLT2 inhibitors are likely to be used for the treatment of heart failure (HF), nonalcoholic steatohepatitis (NASH), chronic kidney disease (CKD), hypertension, obesity etc. SGLT2 inhibitors mitigate numerous complications that may worsen the intensity of diabetes mellitus. Most of the benefits resulted from the use of SGLT2 inhibitors modify the quality of life of person with diabetes towards improvement. But there is also controversy that SGLT2 inhibitors elicit several unwanted effects like cancer, bone fracture, amputation risk etc. However, the standardization of this drugs class for the treatment of new indication can be ensured after thorough investigation with a view to generating sufficient data for better understanding.
Arbutus unedo L., (Ericaceae) inhibits intestinal glucose absorption and improves glucose tolerance in rodents
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Arbutus unedo L., (Ericaceae) is one of the most traditional plants commonly used to treat diabetes in people living in Eastern Morocco region particularly in Taza and Beni Mellal.
The aim of the study was to find if there is a scientific support to the ethnopharmacological relevance use of Arbutus unedo L., roots bark (AU) to treat diabetes.
We studied the effects of crude aqueous extract of AU on intestinal glucose absorption using short-circuit current technique in vitro and oral glucose tolerance test in vivo.
The aqueous extract of AU (10 µg/mL to 1 mg/mL) induced concentration-dependent inhibition of sodium-dependent glucose transport across isolated mouse jejunum. The maximal inhibition was obtained with 1 mg/mL, which exhibited more than 80% of the Phloridzin inhibition with an IC₅₀ close to 216 µg/mL. A 6-week AU ingestion (2 g/(kg day)), improved oral glucose tolerance as efficiently as metformin (300 mg/(kg day)). Arbutus unedo L. and metformin also reduced body weight.
Arbutus unedo L. roots bark aqueous extract directly inhibited the electrogenic intestinal absorption of glucose in vitro. In addition it improved oral glucose tolerance and lowered body weight in rats after chronic oral administration in vivo. These results add a scientific support to the ethnopharmacological relevance use of Arbutus unedo L. roots bark to treat diabetes.
Diabetes and cancer: Pathophysiological fundamentals of a ‘dangerous affair’
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Canagliflozin was shown to be not genotoxic, and in carcinogenicity studies in mice and dogs no pre-neoplastic lesions were detected. Although a 2-year carcinogenicity study in rats showed a higher incidence of pheochromocytomas, Leydig cells tumors and renal tubule tumors, these were attributed to specific features of metabolism regarding carbohydrate malabsorption and calcium homeostasis in rodents and thus not relevant to humans [94]. In a meta-analysis of 46 randomized controlled studies, SGLT2 inhibitors showed no association with overall cancer (OR 1.14, CI-95% 0.96–1.36), even though the statistical analysis for each type of malignancy suggested that the risk of bladder cancer might be increased (OR 3.87, CI-95% 1.48–10.08), especially with empagliflozin (OR 4.49, CI-95% 1.21–16.73) [95].
Diabetes and cancer are worldwide chronic diseases with a major impact on the quality and expectancy of life. Metabolic abnormalities observed during the onset and progression of diabetes may have a critical role on the initiation and progression of carcinogenesis. To date, there are no conclusive data on the mechanisms underlying the relationship between diabetes and any type of human cancer. However, recent evidence suggests that both hyperglycemia and hyperinsulinemia in diabetes could elicit cell damage responses, such as glucotoxicity, lipotoxicity and oxidative stress, which participate in the cell transformation process raising the risk of cancer development. In addition, clinical trials have revealed that several anti-diabetes therapies may potentially affect the risk of cancer though largely undefined mechanisms. In this review, we highlight epidemiological and pathophysiological aspects of diabetes, which may influence cancer initiation and progression.
Renal tubular and adrenal medullary tumors in the 2-year rat study with canagliflozin confirmed to be secondary to carbohydrate (glucose) malabsorption in the 15-month mechanistic rat study
2017, Chemico-Biological Interactions
Citation Excerpt :
In rats at high doses canagliflozin also inhibits the absorption of glucose from the small intestine via the inhibition of SGLT1. Canagliflozin was not genotoxic in a battery of in vitro assays (Ames, mouse lymphoma) and in vivo studies (micronucleus test and comet assay) [1]. In a 2-year rat carcinogenicity study, tumors were observed in the kidney, adrenal glands, and testes [1].
During preclinical development of canagliflozin, an SGLT2 inhibitor, treatment-related pheochromocytomas, renal tubular tumors (RTT), and testicular Leydig cell tumors were reported in the 2-year rat toxicology study.
In a previous 6-month rat mechanistic study, feeding a glucose free diet prevented canagliflozin effects on carbohydrate malabsorption as well as the increase in cell proliferation in adrenal medulla and kidneys, implicating carbohydrate malabsorption as the mechanism for tumor formation.
In this chronic study male Sprague-Dawley rats were dosed orally with canagliflozin at high dose-levels (65 or 100 mg/kg/day) for 15 months and received either a standard diet or a glucose-free diet. Canagliflozin-dosed rats on standard diet showed presence of basophilic renal tubular tumors (6/90) and an increased incidence of adrenal medullary hyperplasia (35/90), which was fully prevented by feeding a glucose-free diet (no RTT's; adrenal medullary hyperplasia in ≤5/90).
These data further confirm that kidney and adrenal medullary tumors in the 2-year rat study were secondary to carbohydrate (glucose) malabsorption and were not due to a direct effect of canagliflozin on these target tissues.

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Carcinogenicity in rats of the SGLT2 inhibitor canagliflozin

Highlights

Abstract

Introduction

Section snippets

Test substance

Animals

Body weight, food consumption, and clinical observations

Discussion

Conclusion

Funding

Conflict of Interest

Transparency Document

Acknowledgements

Chem. Biol. Interact.

Toxicol. Appl. Pharmacol.

Food Chem. Toxicol.

Regul. Toxicol. Pharmacol.

Reprod. Toxicol.

Active sugar transport in health and disease

J. Intern. Med.

Phlorizin: a review

Diabetes Metab. Res. Rev.

Renal glucose transporters: novel targets for hyperglycemia management

Nat. Rev. Nephrol.

SGLT2 inhibition – a novel strategy for diabetes treatment

Nat. Rev. Drug Discov.

Effect of canagliflozin on renal threshold for glucose, glycemia, and body weight in normal and diabetic animal models

PLoS ONE