Effect of Canagliozin on Estimated Fluid Volumes in Patients with Heart Failure and type 2 Diabetes: A Post-hoc Analysis of the CANDLE Trial

Background: In patients with chronic heart failure (CHF) and type 2 diabetes (T2D), inhibition of the sodium-glucose cotransporter-2 (SGLT2) improves cardiorenal outcomes, but the effects of the SGLT2 inhibitor canagliozin on body uid volume and renal function remain to be claried. Methods: This was a post-hoc analysis of 233 patients with CHF and T2D in the CANDLE Trial (UMIN000017669), an investigator-initiated, multi-center, randomized open-label trial that compared the effect of canagliozin (100 mg, n=113) with glimepiride (starting dose: 0.5 mg, n=120) on changes in N-terminal pro-brain natriuretic peptide. The time courses of estimated plasma volume (ePV, calculated with the Straus formula), estimated extracellular volume (eEV, determined by the body surface area), and estimated glomerular ltration rate (eGFR, calculated with the modied Cockcroft formula) were compared between the canagliozin and glimepiride groups at weeks 4, 12, and 24.


Introduction
Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the risk of cardiovascular death or hospital admission for heart failure and prevent kidney disease progression in patients with type 2 diabetes (T2D) [1]. Furthermore, in patients with heart failure (HF), regardless of ejection fraction (EF), SGLT2 inhibitors improve mortality and morbidity and renal outcomes irrespective of the presence of T2D [2][3][4][5].
The mechanisms underlying these effects remain unknown, and reductions in body uid volume and tissue decongestion may contribute. Primarily, SGLT2 inhibitors reduce glucose and sodium reabsorption in the proximal convoluted tubules and lead to glucosuria and natriuresis; therefore, they decrease uid volume but potentially decrease renal function. Indeed, in the Empire HF Renal trial, 12 weeks of treatment with empagli ozin reduced the measured glomerular ltration rate (GFR) compared with placebo in patients with HF, while the estimated circulating plasma volume (ePV) and estimated extracellular uid volume (eEV) were reduced [6]. Furthermore, we reported previously that empagli ozin, compared with placebo, reduced ePV and eEV at the expense of an initial drop in estimated glomerular ltration rate (eGFR) in patients with miscellaneous cardiovascular disease with T2D [7]. However, glucosuria resulting from treatment with SGLT-2 inhibitors may potentially promote electrolyte-free water clearance and increase plasma osmolarity [8-10], and thereby prevent excess reduction of plasma volume while removing interstitial uid in the longer term.
We hypothesized that canagli ozin, as well as empagli ozin, can reduce body uid volume. In the present post-hoc analysis of the CANDLE Trial (UMIN000017669), we aimed to compare the effects of the SGLT-2 inhibitor canagli ozin with those of the sulfonylurea glimepiride on ePV, eEV, and eGFR in the longer term in patients with chronic heart failure (CHF) and T2D.

Study Design and Participants
The present study was a post-hoc analysis of the CANDLE trial (UMIN000017669), the protocol and main results of which were published previously [11,12]. Brie y, the CANDLE trial was an investigator-initiated, open-label, randomized, blinded-endpoint trial carried out at 34 centers in Japan. Key inclusion criteria were T2D under poor or suboptimal control, HF with New York Heart Association (NYHA) functional I-III symptoms, stable condition under guideline-directed HF therapy at least 4 weeks before randomization, and an eGFR greater than 45 mL/min per 1.73 m². Key exclusion criteria were HF with NYHA class IV symptoms and history of cardiovascular disease requiring revascularization within 3 months before randomization. Eligible patients were assessed at a screening visit that included medical history, physical examination, and blood tests. At the baseline visit, patients were randomly assigned (1:1) to treatment with canagli ozin or glimepiride. Treatment assignment was carried out with a web-based program with the minimization method, with biased coin assignment balancing for age (<65, ≥65 years), HbA1c level (<6.5%, ≥6.5%), and left ventricular EF (<40%, ≥40%) at the time of screening. The trial was approved by the institutional review board and independent ethics committees at each site, in compliance with the Declaration of Helsinki and the current legal regulations in Japan.

Estimation of body uid volume
After randomization, patients initiated canagli ozin 100 mg once daily or glimepiride 0.5 mg once daily for 24 weeks. All patients were treated according to the Japanese treatment guidelines for diabetes that allowed increasing the dose of background therapy for diabetes except for SGLT2 inhibitors and sulfonylureas in both groups. Assessment procedures were repeated at weeks 4, 12 and 24 after the baseline visit. Estimated plasma volume was calculated with the Strauss formula according to the data obtained at each visit [13,14].
ΔePV [%]: 100×Hb (base) /Hb (end) ×(1 -Ht (end))/(1 -Ht (base)) − 100 Estimated extracellular volume in mL was determined by the body surface area on the basis of the following formula [15], based on the linear correlation between the volume of distribution of ¹Cr-EDTA (which is the actual extracellular volume) and the body surface area. Statistical analyses Baseline characteristics were described by percentages frequencies for categorical variables and by means with standard deviations for continuous variables. All statistical analyses were done by the intention-to-treat principle. For follow-up ePV, eEV and eGFR values, analyses were performed with linear mixed models with adjustment for the baseline value. The mean values with 95% con dence intervals (CI) at weeks 4, 12 and 24 for both groups were plotted and compared with Wald tests. The consistency of treatment effect was examined across subgroups of age, sex, BMI, systolic blood pressure (SBP), HbA1c, eGFR and EF at baseline. All statistical analyses were carried out at the two-sided signi cance level of 0.05, and no adjustment for multiplicity was considered in this post-hoc sub-analysis. R software version 3.6.3 (R Foundation for Statistical Computing) was used to perform the statistical analyses.
Time course of ePV, eEV, eGFR and SBP Changes in ePV, eEV, eGFR and SBP from baseline to weeks 4, 12 and 24 are shown in Tables 2 and 3 and plotted in Figure 1.

Comparison between canagli ozin and glimepiride effects on body uid volume
In the canagli ozin group, ePV and eEV decreased gradually until week 12 and stayed lower at week 24 than in the glimepiride group. eGFR became signi cantly lower at week 4, but the difference became nonsigni cant at 12 weeks. (Figure 1, Table 3) The effects of canagli ozin versus glimepiride on ePV, eEV and eGFR observed in the overall population were relatively consistent in various patient subgroups. Effect of canagli ozin treatment on body uid volume Diuretic treatment-related weight loss and negative uid balance are associated with improvements in symptoms [17][18][19][20] and recently, for optimal decongestion, discrimination of the interstitial and intravascular compartments has been proposed. Body uid is divided into intra-and extracellular compartments, and the extracellular compartment is divided into intra-or extravascular volume. A large intravascular volume can cause organ congestion in patients with HF, and a small intravascular volume can cause dehydration, leading to low organ perfusion in CHF. On the other hand, the extravascular volume is mainly interstitial and of unknown signi cance in organ dysfunction in CHF. Diuretics reduce the uid volume from the extracellular compartment. Initially, they reduce mainly the intravascular volume rapidly, leading to lower hydrostatic pressure and subsequent uid removal from the interstitial volume [21,22]. However, eventually, the response to diuretic treatment predominately re ects uid loss from the interstitial volume with minimal change in intravascular volume [23]. Recently, it was reported that the degree of interstitial volume reduction was not associated with patient prognosis [23], suggesting that the intravascular volume is more a driver of clinical outcomes than even large changes in the interstitial volume [13,24].
In this study, canagli ozin treatment of patients with CHF and T2D decreased the eEV as well as the ePV, without a rebound increase over a long period of time. This nding has already been shown with another SGLT2 inhibitor [6,7]. Consistent results support a class effect of SGLT2 inhibitors on reducing the intravascular volume. Reduction in the intravascular uid volume, which decreases the ventricular lling pressure and cardiac workload, is a relevant mechanism to explain the reduction in HF risk with SGLT2 inhibitors [25].

Temporal relationship between eGFR and body uid volume
The mechanisms of the renoprotective effects of SGLT2 inhibitors remain to be elucidated. Lowering intraglomerular hypertension by tubuloglomerular feedback [26-28], reduction of tubular workload [29], anti-in ammation and anti-brosis [30, 31] have been reported as major mechanisms. In addition, from our results, we hypothesize that maintenance of optimal intravascular volume results in protection of renal perfusion. In the present study, both ePV and eEV decreased gradually and took more than 4 weeks to become statistically signi cant; furthermore, ePV decreased until week 12 and stopped decreasing. In contrast, eEV continued to decrease until week 24. The body uid volume response to canagli ozin is clearly slower than that for loop diuretics, with which strong and rapid diuresis can lead to poor renal perfusion and potential nephrotoxicity [32]. However, this time course of ePV may imply avoidance of excessive decongestion for renal perfusion and maintenance of optimal intravascular volume throughout the 24-week observation period. Moreover, recent studies have identi ed important differences between SGLT2 inhibitors and loop diuretics. Mathematical model analyses of dapagli ozin and bumetanide have suggested that dapagli ozin produces weaker natriuretic and diuretic effects than bumetanide, and the reduction in blood volume compared with interstitial volume was smaller with dapagli ozin [8]. The ability to provide selective interstitial uid reduction may be a unique feature of SGLT2 inhibitors, and maintenance of arterial lling may suppress neurohormonal activation [33, 34] and lead to renoprotective effects. Estimation of the intravascular volume by ePV may re ect renal perfusion and lead to safe clinical management and better understanding of the mechanisms underlying the renoprotective effects of SGLT2 inhibitors in HF treatment.

Limitations
This analysis has several limitations. First, it was conducted post hoc to explore the mechanism underlying the cardiorenal protective effects of canagli ozin. The original study was not speci cally designed to examine changes in body uid distribution, nor did it attempt to balance or stratify patients by different types of HF.
Second, the PV and EV changes were not measured directly, but instead were estimated by widely accepted formulae using Hct, Hb and body weight, because these were validated by comparison with a radiolabeled gold standard method in patients with HF and diabetes [14,35].
Despite these limitations, the time course of ePV and eEV were theoretically consistent with the change in eGFR, and furthermore with the cardiovascular bene ts reported in larger clinical trials. Further study with direct measurement of plasma volume and extracellular volume appears warranted.
Declarations outcomes in type 2 diabetes: a systematic review and meta-analysis of cardiovascular outcome trials. Lancet (London, England)2019, 393(10166):31-39.  NT-proBNP, N-terminal pro-brain natriuretic peptide; NYHA, New York Heart Association. Table 2 Changes from baseline to weeks 4, 12 and 24 in ePV, eEV, eGFR and SBP   Figure 1 Changes in ePV, eEV, eGFR, and SBP after administration of canagli ozin and glimepiride A. Estimated plasma volume B. Estimated extracellular volume C. Estimated glomerular ltration rate D. Systolic blood pressure Data are expressed as mean (95% con dence interval).