The efficacy and safety of roxadustat for the treatment of anemia in non-dialysis dependent chronic kidney disease patients: An updated systematic review and meta-analysis of randomized clinical trials.

BACKGROUND: Roxadustat (ROX) is a new medication for anemia as a complication of chronic kidney disease (CKD). Our meta-analysis aims to evaluate the efficacy and safety of ROX, especially on the cardiovascular risks, for anemia in NDD-CKD patients.
METHODS: Electronic databases were searched systematically from inception to July 2021 to look for randomized control trials (RCTs) that evaluated ROX NDD-CKD patients. Hemoglobin level and iron utilization parameters, including ferritin, serum iron, transferrin saturation (TSAT), total iron-binding capacity (TIBC), transferrin, and hepcidin were analyzed for efficacy. Pooled risk ratios (RRs) and standardized mean differences (SMDs) were calculated and presented with their 95% confidential intervals (CIs).
RESULTS: Nine RCTs included a total of 3,175 patients in the ROX group and 2,446 patients in the control group. When compared the control group, ROX increased Hb level significantly (SMD: 1.65; 95% CI: 1.08, 2.22; P< 0.00001) and improved iron utilization parameters by decreasing ferritin (SMD: -0.32; 95% CI: -0.51, -0.14; P = 0.0006), TSAT (SMD: -0.19; 95% CI: -0.32, -0.07; P = 0.003), and hepcidin (SMD: -0.74; 95% CI: -1.09, -0.39; P< 0.0001) and increasing TIBC (SMD: 0.99; 95% CI: 0.76, 1.22; P< 0.00001) and transferrin (SMD: 1.20; 95% CI: 0.70, 1.71; P< 0.00001). As for safety, ROX was associated with higher serious adverse effects (RR: 1.07; 95% CI: 1.01, 1.13; P = 0.01), deep venous thrombosis (DVT) (RR: 3.80; 95% CI: 1.5, 9.64; P = 0.08), and hypertension (HTN) (RR: 1.37; 95% CI: 1.13, 1.65; P = 0.001).
CONCLUSION: We concluded that ROX increased Hb level and improved iron utilization parameters in NDD-CKD patients, but ROX was associated with higher serious adverse effects, especially DVT and HTN. Our results support the use of ROX for NDD-CKD patients with anemia. However, higher-quality RCTs are still needed to ensure its safety and risk of thrombosis.

Introduction

Anemia is common in non-dialysis chronic kidney disease (ND-CKD) patients and, as the glomerular filtration rate (GFR) decreases, the prevalence increases [1, 2]. The prevalence of anemia in patients with stage 1 CKD is 8.4% compared to 53.4% at stage 5 CKD [3], and it is associated with increased hospitalization, mortality, and a decrease in the quality of life [4, 5]. Therefore, all patients require anemia screening when assessing CKD for the first time and before administering any therapy, and the patient should be monitored frequently for the therapy response [6]. The anemia in CKD patients is multifactorial [7]. Erythropoietin (EPO) is a hormone that stimulates red blood cell synthesis, and it is produced by renal EPO-producing cells located in the interstitium of the outer medulla and cortex [8]. CKD is characterized by interstitial fibrosis leading to EPO insufficiency [9]. Other factors include iron deficiency, decreased oxygen sensing, and accumulation of uremic toxins [10].

The treatment of anemia in ND-CKD patients depends on the anemia severity, and the main treatment options include iron and erythropoiesis-stimulating agents (ESAs) [5], but it is associated with increased risk of serious cardiovascular events, myocardial infarction (MI), stroke, and venous thromboembolism that limit its usage [10-12].

Roxadustat (ROX) is a new medication that reversibly inhibits hypoxia-inducible factor prolyl hydroxylase (HIF-PH) enzymes, leading to improved oxygen sensing and increasing hemoglobin levels. ROX showed efficacy and safety in NDD-CKD patients and had the first global approval in China to treat anemia in NDD-CKD patients [13].

Our study aims to conduct an updated meta-analysis of randomized clinical trials (RCTs) by analyzing previously published RCTs besides the most recent RCTs Akizawa et al. [14], Barratt et al. [15], and Shutov et al. [16]. We will investigate the efficacy and safety of ROX to placebo of ESAs in the treatment of anemia in NDD-CKD patients. Our study will help internists and nephrologists decide whether ROX should be considered in managing anemia in NDD-CKD patients.

Methods

Data sources and search strategy

We followed Cochrane Handbook for Systematic Reviews of Interventions and Preferred Reporting for Systematic Review and Meta-Analysis (PRISMA) to conduct this systematic review and meta-analysis [17, 18]. We registered our meta-analysis at OSF Registries with DOI 10.17605/OSF.IO/WGZ6C. Systematic research of PubMed, EMBASE, Scopus, Web of Science, Cochrane Central, and Google Scholar was searched systematically from inception through July 2021 to include citations on non-HD CKD patients treated with ROX for anemia. The following search terms were used (Roxadustat OR ASP1517 OR FG4592 OR "FG-4592") AND (kidney OR renal) AND (Anemia), and it varies depending on the database (S1 Table). The related articles’ feature [19] was used to include any related articles, and the references of the included studies were manually reviewed to include any relevant citation. EndNote [20] was used to save the search result, and the result was transferred to Covidence for screening [21]. A further manual search was done on November 1, 2021, to look for recently published articles.

Study selection and eligibility criteria

We included studies that met the following criteria: study design was RCTs; written English text; the target population was patients CKD and not on HD; the intervention was ROX and compared placebo or any other medications; primary outcomes changed in hemoglobin level and iron parameters and studies reported the outcomes of interest were included. We excluded the observational studies, studies that did not report a comparator group, and non-randomized studies. Two independent reviewers (JS and KSA) completed the title, abstract, and full-text screening. Any conflict between the authors was resolved by a third author (BA).

Data extraction

The data from the included studies were extracted independently by three reviewers (JS, KSA, and NAE). The consensus was reached in case of any inconsistency by the fourth author (BA). Each included RCT was abstracted for the first author, published date, country, study design, phase, study period, number, age, gender of patients, and ROX dose.

Risk of bias assessment

Two Reviewer (KSA and NAE) performed the risk of bias assessment independently using the revised Cochrane risk of bias 2 tool (Rob 2) [22] to evaluate the randomization process, deviations from the intended interventions, missing outcome data, measurement of the outcome, selection of the reported results, and overall risk of bias. The overall grade of each aspect was measured as low risk, high risk, or some concerns.

Outcomes of interest

The primary outcomes are changes in hemoglobin (Hb) level and iron utilization parameters, including ferritin, serum iron, transferrin saturation (TSAT), Total iron-binding capacity (TIBC), transferrin, and hepcidin. Secondary outcomes are serious adverse events, treatment-emergent adverse effects (TEAEs), and cardiovascular-related adverse effects; hypertension (HTN), hypertensive crisis, pulmonary edema, heart failure, coronary artery diseases, Myocardial infarction (MI), and deep venous thrombosis (DV).

Statistical analysis

Meta-analyses of all outcomes were performed using RevMan manager v5.3 [23] by Two authors (KSA and NAE) and reviewed by BA. Risk ratios (RRs) were used for the dichotomous outcomes and the standardized mean difference (SMD) for the continuous outcome, and both presented it along with the corresponding 95% confidence interval (CI). Data were analyzed using the Mantel–Haenszel method. A P-value less than 0.05 is considered significant. All outcomes were calculated according to the fixed-effects model (in the absence of significant heterogeneity) and the random-effects model (in the presence of significant heterogeneity). Statistical heterogeneity between trials was evaluated using the Cochran Q test and measured using I2 statistics. I2> 50% indicates significant heterogeneity across included RCTs [24]. The risk of publication bias was not performed because we included less than ten RCTs [25]. We excluded one study at a time and repeated the analysis to perform the sensitivity analysis by removing one study at a time to assess the impact of each study on the overall study effect on the Hb level. Subgroup analysis was carried out to investigate the impact of the trial phase and the different control groups (ROX Vs. ESA and ROX Vs. placebo) on the Hb level.

We used Comprehensive Meta-Analysis (CMA) [26] software to perform meta-regression to study the effect of rescue therapy or iron supplementation on the mean change of hemoglobin level. Standardized differences in means and the total of ROX versus control were tabulated with the status of rescue therapy and iron supplement; allowed or prohibited. A scatter plot was created for two models: rescue therapy and iron supplement.

Results

Search results and study selection

From a pool of 908 potentially relevant articles, we chose 62 for full full-text review according to our inclusion and exclusion criteria. Thus, a total of nine RCTs were included in our systematic review and meta-analysis [14–16, 27–32]. The process of inclusion and exclusion with the reasons for exclusion was shown in the PRISMA flow diagram Fig 1.

Fig 1

PRISMA 2020 flow diagram for updated systematic reviews, which included searches of databases, registers, and other sources.

Characteristics of included studies

The main characteristics of the studies included are presented in Table 1. Six RCTs were double-blinded [16, 27–30, 32], one was single-blinded [31], and two were open-label [14, 15]. A total of six RCTs were phase 3 [14–16, 27, 28, 30], and three were phase 2 [29, 31, 32]. Two RCTs compared ROX to darbepoetin alfa [14, 15], and the rest of the RCTs compared ROX to placebo. The ROX dose ranged from 50 mg to 100 mg three times a week, and two studies used the dose according to the weight ranging from 0.7 to 2.25 mg/kg [29, 31]. The study duration ranged from 8 weeks to 104 weeks. The mean age of the included patient was 62 ± 14.8 years, and 43.5% were male. The primary causes of the CKD and the rest of the patients’ demographics and baseline characteristics were summarized in Table 2.

Table 1

Characteristics of the included studies.

Author, year	Location (sites number)	Study design	Study period	Groups	Number of patients	Age in years, Mean ± SD	Male ratio, %	Roxadustat dose	Study duration	Phase
Akizawa et al. 2019 [32]	Japan (32)	Double-blinded RCT	2013–2015	Roxadustat	80	64.4 ± 8.7	48.80%	(50 mg,70 mg, and 100 mg) TIW	24 weeks	2
				Placebo	27	61.9 ± 10.6	40.70%
Akizawa et al. 2021 [14]	Japan (71)	Open-label RCT	2017–2020	Roxadustat	131	68.9 ± 11.6	63.40%	(70mg or 100mg) TIW	52 weeks	3
				DA	131	70.9 ± 10.2	57.30%
Barratt et al. 2021 [15]	Europe (200)	Open-label RCT	2014–2018	Roxadustat	323	66.8 ± 13.6	44.90%	(Weight 45 to 70 kg, 70 mg; weight >70 to 160 kg, 100 mg) TIW	104 weeks	3
				DA	293	65.7 ± 14.4	44%
Besarab et al. 2015 [33]	United States (29)	Single-blind RCT	2008–2010	Roxadustat	88	64	37.50%	(0.7, 1, 1.5 or 2 mg/kg) BIW or TIW	4 weeks	2a
				Placebo	28	68.6	57.10%
Chen et al. 2017 [29]	China (11)	Double-blinded RCT	2011–2012	Roxadustat	61	48.9 ± 13.8	29.50%	low (1.1–1.75 mg/kg) or high (1.50–2.25 mg/kg) TIW	8 weeks	2
				Placebo	30	51.4 ± 11.9	26.70%
Chen et al. 2019 [30]	China (29)	Double-blinded RCT	2015–2016	Roxadustat	101	54.7 ± 13.3	36%	(Weight 40 to <60 kg, 70mg; weight ≥60 kg, 100mg) TIW	8 weeks	3
				Placebo	51	53.2 ± 13.1	39%
Coyne et al. 2021 [28]	United States, South America, Australia, New Zealand, and Asia (163)	Double-blinded RCT	2012–2018	Roxadustat	616	64.9 ± 12.6	39.10%	(Weight 45 to <70 kg, 70mg; weight ≥70 kg, 100mg) TIW	28–52 weeks	3
				Placebo	306	64.8 ± 13.2	43.50%
Fishbane et al. 2021 [34]	25 Countries (385)	Double-blinded RCT	2014–2018	Roxadustat	1384	60.9 ± 14.7	40.80%	70mg TIW	28–52 weeks	3
				Placebo	1377	62.4 ± 14.1	43.80%
Shutov et al. 2021 [16]	Different countries, mainly from Europe (138)	Double-blinded RCT	2013–2017	Roxadustat	391	58.25 ± 19.91	43.20%	(Weight 45 to ≤70 kg, 70mg; weight >70 to ≤ 160 kg, 100mg) TIW	52–104 weeks	3
				Placebo	203	60.5 ± 18.46	48.80%

Continuous variables are expressed in mean ± standard deviation.

RCT = randomized control trial; DA = darbepoetin alfa; TIW = three times a week; DIW = two times a week; SD: standard deviation.

Table 2

The patient demographics and baseline characteristics.

Author, Year	Groups	Race, n (%)	Bodyweight (kg)	eGFRc (mL/min/1.73 m2)	CKD stage
Akizawa et al. 2019 [32]	Roxadustat	Japanese 80 (100%)	59.07 ± 9.83	16.3 ± 7.8	2 to 5
	Placebo	Japanese 27 (100%)	60.17 ± 8.72	16.3 ± 8.5
Akizawa et al. 2021 [14]	Roxadustat	Japanese 131 (100%)	N/A	17.9 ± 8.2	N/A
	DA	Japanese 131 (100%)	N/A	18.2 ± 8.8
Barratt et al. 2021 [15]	Roxadustat	White 306 (94.7%), Black 8 (2.5%), Asian 9 (2.8%)	76.90 ± 16.33	20.31 ± 11.49	3 to 5
	DA	White 281 (95.9%), Black 2 (0.7%), Asian 10 (3.4%)	78.39 ± 17.68	20.34 ± 10.73
Besarab et al. 2015 [33]	Roxadustat	White 49 (55.7%), Black 34 (38.6%), Asian 2 (2.3%)	N/A	34.3 ± 12.7	3 to 4
	Placebo	White 15 (53.6%), black 11 (39.3%), Asian 2 (7.1%)	N/A	31.4 ± 12.4
Chen et al. 2017 [29]	Roxadustat	Chinese 61 (100%)	57.4 ± 11	19.4 ± 9.5	1 to 4
	Placebo	Chinese 30 (100%)	56.9 ±10.3	23.0 ±13.4
Chen et al. 2019 [30]	Roxadustat	N/A	N/A	16.5 ± 8	3 to 5
	Placebo	N/A	N/A	14.5 ± 7.6
Coyne et al. 2021 [28]	Roxadustat	N/A	N/A	21.9 ± 11.5	3 to 5
	Placebo	N/A	N/A	22.4 ± 11.4
Fishbane et al. 2021 [34]	Roxadustat	White 623 (45%), Black 112 (8.1%), Asian 544 (39.3%), American Indian 24 (1.7%)	69.9 ± 18.5	19.7 ± 11.7	3 to 5
	Placebo	White 611 (44.4%), Black 115(8.4%), Asian 538 (39.1%), American Indian 29 (2.1%), Native Hawaiian 2 (0.1%)	70.6 ± 18.8	20 ± 11.7
Shutov et al. 2021 [16]	Roxadustat	White 335 (85.7%), Black 10 (2.6%), Asian 9 (2.3%)	73.86 ± 16.49	16.5 ± 10.2	3 to 5
	Placebo	White 182 (89.7%), Black 3 (1.5%)	76.50 ± 16.51	17.2 ± 11.7

Continuous variables are expressed in mean ± standard deviation.

DA = darbepoetin alfa; eGFR = estimated glomerular filtration rate; CKD = chronic kidney disease; CPN = chronic pyelonephritis; PKD = Polycystic kidney disease; N/A = not applicable.

The overall risk of bias was judged as some concerns for three studies [27-29] and as high risk in six studies [14–16, 30–32]. All studies were judged with some concerns on the randomization process, except two had a low risk of bias [14, 32], and one was a high risk of bias [15]. All studies were judged with a low risk of bias for deviations from intended interventions and measurement of the outcome, except three studies have some concerns [14, 15, 31]. Six studies were judged low risk for the missing outcome data [14, 15, 27–29, 32] and three with some concerns [16, 30, 31]. Finally, all studies were judged with a low risk of bias for selective reporting, except two studies were judged as high risk of bias [31, 32]. Fig 2 demonstrated the risk of bias summary and graph, and the detailed assessment of the risk bias will be found in S1 File.

Fig 2

Risk of bias assessment.

A: Risk of bias summary: review authors’ judgments about each risk of bias item for each included study. The items are scored (+) low risk; (-) high risk; (?) some concerns. B Risk of bias graph: review authors’ judgments about each risk of bias item presented as percentages across all included studies.

Primary endpoints

Hemoglobin level

ROX showed an increase in Hb level when compared to control (SMD: 1.65; 95% CI: 1.08, 2.22; P< 0.00001) (Fig 3, Forest plot A). Sensitivity analysis by omitting one study at a time showed a consistent result of ROX on Hb (S2 Table). Subgroup analysis to investigate the effect of the trial phase on the Hb level showed a similar result. Three RCTs were in Phase 2 (SMD: 3.01; 95% CI: 0.91, 5.11; P< 0.005) [29, 31, 32] and six RCTs were in phase three (SMD: 1.12; 95% CI: 0.49, 1.75; P< 0.0005) [14–16, 27, 28, 30] (S1 Fig). However, when we compared ROX to placebo, there was an increase in Hb level (SMD: 2.11; 95% CI: 1.58, 2.63; P< 0.00001), but when we compared ROX to darbepoetin alfa (DA), there was no difference between the two groups (SMD: -0.00; 95% CI: -0.14, 0.14; P< 1.00) (S2 Fig). There were only two RCTs that compared the effect of ROX to DA [14, 15], so more RCTs are needed to confirm the result.

Fig 3

Forest plot of the primary outcomes.

A: hemoglobin level; B: ferritin; C: TSAT; D: hepcidin; E: TIBS; F: serum iron; G: transferrin; CI: confidence interval; M-H: Mantel-Haenszel; df: degrees of freedom; I2, I-squared.

Iron parameters

When compared to the control group, ROX showed decrease in ferritin level (SMD: -0.32; 95% CI: -0.51, -0.14; P = 0.0006) (Fig 3, Forest plot B), TSAT (SMD: -0.19; 95% CI: -0.32, -0.07; P = 0.003) (Fig 3, Forest plot C), hepcidin (SMD: -0.74; 95% CI: -1.09, -0.39; P< 0.0001) (Fig 3, Forest plot D). And ROX showed increase in TIBC (SMD: 0.99; 95% CI: 0.76, 1.22; P< 0.00001) (Fig 3, Forest plot E), transferrin (SMD: 1.20; 95% CI: 0.70, 1.71; P< 0.00001) (Fig 3, Forest plot F). There was no difference between ROX and the control group regarding serum iron level (SMD: 0.53; 95% CI: -0.36, 1.42; P = 0.25) (Fig 3, Forest plot G).

Secondary endpoints

The ROX group showed higher serious adverse effects when compared to the control group (RR: 1.07; 95% CI: 1.01, 1.13; P = 0.01) (Fig 4, Forest plot A). Subgroup analysis comparing ROX to placebo showed a similar result (RR: 1.07; 95% CI: 1.01, 1.13; P = 0.03), However, subgroup analysis comparing ROX to DA showed no difference between the two groups (RR: 1.07; 95% CI: 0.95, 1.21; P = 0.26) (S3 Fig). There was no difference between both groups regarding the TEAEs (RR: 1.02; 95% CI: -1.00, 1.04; P = 0.08) (Fig 4, Forest plot B).

Fig 4

Forest plot of the secondary outcomes.

A: serious adverse effect; B; treatment-emergent adverse effects (TEAE); C: hypertension; D: deep venous thrombosis; CI: confidence interval; M-H: Mantel-Haenszel; df: degrees of freedom; I2, I-squared.

Cardiovascular-related adverse effect; Our results did not show a significant difference in the risk ratio of hypertensive crisis, pulmonary edema, heart failure, coronary artery diseases, and MI (S4–S8 Figs). Meanwhile, significant risk was associated with ROX for HTN and (RR: 1.37; 95% CI: 1.13, 1.65; P = 0.001) (Fig 4, Forest plot C) and DVT (RR: 3.80; 95% CI: 1.5, 9.64; P = 0.08) (Fig 4, Forest plot D) respectively.

Meta-regression

Meta-regression on eight studies showed that resume therapy and iron supplementation led to decrease in the mean difference between the ROX and control group; (SMD: 1.65; 95% CI: 1.08, 2.22; P< 0.00001) before meta-regression versus (SMD: 2.78; 95% CI: .89,4.669; P = 0.009) after regression. A Scatter plot was created for better visualization (S9 and S10 Figs).

Discussion

In our updated meta-analysis of RCTs, we included 5359 NDD-CKD patients to evaluate the safety and efficacy of ROX compared to placebo or ESA. We concluded that ROX increased Hb level and improved iron utilization parameters by decreasing ferritin, TSAT, hepcidin, and increasing TIBC and transferrin. Those results can be contributed by the effect of ROX on HIF. Hypoxia-inducible factor (HIF) is a transcription factor protein that participates in iron homeostasis and regulates the expression of genes that stimulate erythropoiesis. It consists of an alpha-subunit (HIF- α) and a beta-subunit (HIF- β). HIF- α is oxygen-sensitive and induced by hypoxia, while HIF- β is a constitutive component [7]. Therefore, when the body’s oxygen content is normal, the Hypoxia-inducible factor prolyl hydroxylase (HIF-PH) will increase the breakdown of HIF [35]. Conversely, reduced oxygen transport in anemia induces tissue hypoxia, which stimulates erythropoietin (EPO) production through activation of the HIF system [36]. Erythropoietin induces erythropoiesis by stimulating the division and differentiation of erythroid progenitor cells by activating several signaling pathways, including Janus kinase /signal transducer and activator of transcription 5, phosphatidylinositol 3-kinase /protein kinase B, and Ras-Raf-MEK-ERK pathways [37].

Roxadustat, also known as FG-4592 or ASP1517, is a HIF- prolyl hydroxylase inhibitor and induces EPO production. ROX is an oral medication with peak concentration achieved in one to three hours, with a mean half-life of 12 to 14 hours [38]. ROX increases the hemoglobin level in a dose-dependent manner. RCTs studies the effect of different doses of ROX; Chen et al. reported that ROX showed a dose-response effect on Hb levels. Hg level increase ≥ 1 g/dl from baseline was seen in 80.0% of the patients on low dose regimen (1.1–1.75 mg/kg) compared to 87.1% of the patients with high dose regimen (1.50–2.25 mg/kg) [29]. Akizawa et al. used ROX 50 mg, 70 mg, and 100 mg TIW and reported mean (SD) rate of rise of + 0.200 (5), + 0.453 (5), and + 0.570 (5) for the Hb over the first 6 weeks [32]. Similar findings reported by Besarab et al., the Hb response ranged from 30% in the 0.7 mg/kg two times a week group to 100% in the 2.0 mg/kg two times a week and three times a week groups [31].

ROX improves iron utilization parameters by decreasing ferritin, TSAT, and hepcidin and increasing TIBC and transferrin, thus enhancing the utilization of iron in the body; this may lead to iron deficiency. Therefore, it’s advisable to receive proper iron supplementation during the treatment period to avoid any adverse event. ROX was associated with higher serious side effects when compared to placebo. Fishbone et al. reported the most common adverse effect of ROX was end-stage kidney disease, urinary tract infection, pneumonia, and HTN [27]. Our meta-analysis result showed no difference regarding the TEAEs between ROX and placebo or DA. The pooled analysis of global phase III study by FibroGen and AstraZeneca, including 4270 NDD-CKD patients showed comparable risks of a major adverse cardiovascular event (MACE) (HR = 1.08, 95%CI 0.94,1.24), MACE + unstable angina and heart failure requiring hospitalization (HR = 1.04, 95% CI 0.91,1.18) in patients received ROX to the patients received placebo [39]. Those results were consistent with Provenzano et al., who reported that there were no increased risks of MACE (HR, 1.10; 95% CI, 0.96 to 1.27), MACE+ (HR, 1.07; 95% CI, 0.94 to 1.21), and all-cause mortality (HR, 1.08; 95% CI, 0.93 to 1.26) between ROX and placebo groups [40]. Our results showed a significant risk of HTN and DVT in ROX groups which warrant close monitoring. When we compared ROX to DA, there was no difference between the two drugs regarding the serious adverse effect, but only two RCTs were compared ROX to DA in NDD-CKD patients [14, 15].

The previous meta-analysis of RCTs by Tang et al. [41] included eight RCTs with a total of 5,379 NDD-CKD patients. They concluded that ROX was associated with increased hemoglobin level weighted mean difference {WMD}: 1.36 g/dL; 95% CI: 0.90, 1.82; p < 0.00001, transferrin level (WMD: 0.6 g/L; 95% CI: 0.24, 0.95; p < 0.0009), and TIBC level (WMD: 59.90 μg/dL; 95% CI: 38.85, 80.96; <0.00001). In addition, they reported that ROX lowered the hepcidin level (WMD: −51.31 ng/ml; 95% CI: −67.88, −28.12; p <0.00001) and the ferritin and TAST levels in NDD-CKD patients, and these results were almost similar to other previous systematic reviews [42-44]. Also, Tang et al. [41] reported that there is no difference between the treatment-emergent adverse events (TEAEs) of ROX and ESAs or placebo except for serious TEAEs, which was higher in the ROX group (OR: 1.15; 95% CI: 1.02–1.29; p < 0.02). Tang et al. published their article before the release of Barratt et al. [15], limiting their ability to assess and evaluate the article. We used standardized mean differences for outcomes with different units from the RCTs that used different scales to standardize the outcomes. Even though we added one more study [14] in our included studies, our analysis has a higher number of patients and studies in different outcomes showing our more detailed and focused analysis on NDD-CKD only patients. Liu et al. reported similar results regarding TEAEs as they reported no significant difference among different study groups, but they found an increased risk of developing hyperkalemia in the ROX group [42]. We added an analysis for safety with focusing on cardiovascular adverse effects. We were able to do a detailed meta-analysis with all possible shared outcomes among the included RCTs, detailed sensitivity, subgroup analyses, and meta-regression for concomitant iron supplementations.

The limitation of this study is as follows. First, the overall risk of bias was high in six studies and with some concerns in the other three RCTs, and it is likely due to the lack of a clear randomization process in most of the studies. Therefore, high-quality RCTs are needed in the coming future. Currently, there is an ongoing phase 4 trial assessing the effect ROX versus recombinant human erythropoietin on patients with anemia and CKD (NCT04655027). Second, a high level of heterogeneity detected between the RCTs limits the generalization of our results. The high level of heterogeneity is likely due to different ROX doses and populations. Also, only included RCTs were few in some subgroup analyses, which considered a limitation to our analysis. Third, the included RCTs were sponsored by pharmaceutical companies, and data analysis might be subject to some bias. Fourth, the duration of the included RCTs was short, and they reported only short terms results and could not assess the long-term efficacy and adverse effects of ROX.

In contrast, our study has some strengths. Firstly, we included three RCTs that were recently published. Secondly, we did a detailed meta-analysis and focused on NDD-CKD patients to decrease the potential sources of heterogeneity and bias.

Conclusions

Our review included nine RCTs to assess the effect of ROX on NDD-CKD patients with anemia. We conclude that ROX was associated with increased Hb level and improved iron utilization parameters by decreasing ferritin, TSAT, and hepcidin and increasing TIBC and transferrin. In addition, ROX was associated with higher serious adverse effects, HTN, DVT when compared to placebo. However, higher-quality RCTs are still needed to confirm the results of our review.

PRISMA 2020 checklist.

Preferred reporting items for systematic review and meta-analysis.

(DOCX)

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Forest plot of the effect of trial phase on hemoglobin level.

(DOCX)

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Forest plot of the effect of control arm type on hemoglobin level.

(DOCX)

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Forest plot of the effect of control arm type on serious adverse effects.

(DOCX)

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Forest plot of comparison: 4 Cardiovascular side effects, outcome: 4.2 Hypertensive crisis.

(DOCX)

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Forest plot of comparison: 4 Cardiovascular side effects, outcome: 4.3 Pulmonary edema.

(DOCX)

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Forest plot of comparison: 4 Cardiovascular side effects, outcome: 4.4 Heart failure.

(DOCX)

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Forest plot of comparison: 4 Cardiovascular side effects, outcome: 4.5 Coronary artery disease.

(DOCX)

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Forest plot of comparison: 4 Cardiovascular side effects, outcome: 4.6 Myocardial infarction.

(DOCX)

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Regression of standardized difference in means on rescue.

(DOCX)

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Regression of standardized difference in means on iron.

(DOCX)

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Search terms and results in different databases.

(DOCX)

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Meta-analysis of the primary outcomes and sensitivity analysis.

(DOCX)

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The detailed assessment of the risk bias.

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6 Jan 2022

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this work, the Authors investigated by a systematic review and meta-analysis the effects and the safety of Roxadustat (ROX) in CKD patients not on dialysis therapy.

Comments

- Some further meta-analyses were recently published (Wang et al., Ann Palliat Med 2021, 10:4736–46; Qie et al., Int Urol Nephrol 2021, 53:985–97; Zhang et al., Aging 2021, 13:17914–29; Liu et al., Front Med 2021, 8: 724456) and should be added to the article.

- The Authors report in the present work higher serious adverse events, hypertension, and deep venous thrombosis in ROX treated patients. By contrast, a recent meta-analysis (Liu, see above) showed that total adverse events were not significantly different between ROX and placebo as confirmed by trial sequential analysis, with increased risk of hyperkalemia events. A comment is welcomed.

- Three phase 2 trials were included: may this decrease the level of evidence?

- How were managed studies reporting data for more than one ROX dose?

- The Authors report improved iron utilization parameters upon ROX treatment including decreases in ferritin and transferrin saturation and increases in TIBG and transferrin. These changes however also indicate a decrease in body’s iron storage and available iron, and suggest that patients were not receiving appropriate iron therapy for body iron requirement during ROX treatment. It appears that a stranded iron application is necessary in future studies to better characterize the effects of ROX on iron metabolism. Authors would comment on this issue.

Reviewer #2: Abdelazeem and Colleagues, with this systematic review and meta-analysis, tried to extrapolate from the randomized studies available the efficacy and safety of Roxadustat on change in Hemoglobin and related parameters as well as on cardiovascular risk. The study is of interest and statistical analysis is overall of good quality. Moreover, this is an important topic from a clinical research perspective. However, I have several concerns to share with Authors:

- Abstract: I would suggest Authors to change “efficacy and safety of ROX, especially on the cardiovascular system” into “efficacy and safety of ROX, especially on cardiovascular endpoints/risk”. The word system is too general. Moreover, please change “hat evaluated ROX NDD- CKD patients” with “hat evaluated ROX in NDD-CKD patients”;

- English language should be improved throughout the manuscript.

- Introduction is clearly written, and the aim is well depicted in my opinion.

- Methodology is overall accurate in term of study selection and statistical tools used. However, I have a major concern namely the fact that studies were “intervention was ROX compared placebo or any other medications” were included in the meta-analysis. In this way Authors compared for example ROX with darbepoetin or ROX against no ESA. This can introduce a bias if the primary endpoint is the change in Hb levels.

- Please also improve the quality of figures. Most of them are not clearly interpretable in the current form.

- Some subgroup analyses/meta-analyses (for instance panel D of figure 4) included less then 5 studies and this confers low power to statistical finding. Please clarify this in the limitations if possible.

**********

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Reviewer #1: No

Reviewer #2: No

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27 Jan 2022

Mostafa Eltobgy, MD

The Ohio State University

281 W Lane Ave, Columbus, OH 43210

Tel: 551-227-6556

E-mail: eltobgy.2@osu.edu

January 27th, 2021

Dear Editor-in-Chief,

We submit for the revision for our manuscript entitled “The efficacy and safety of roxadustat for the treatment of anemia in non-dialysis dependent chronic kidney disease patients: an updated systematic review and meta-analysis of randomized clinical trials” with ID PONE-D-21-35500 for consideration in your journal.

Please see the reply to each of the reviewers’ comments below.

Journal Requirements:

When submitting your revision, we need you to adso much ess these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf

and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Reply: Thank you so much for your comments. We ensured that our manuscript met PLOS ONE's style requirements

2. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information:

http://journals.plos.org/plosone/s/supporting-information

Reply: Thank you so much for your comments. We add captions for the Supporting Information files at the end of the manuscript and update any in-text citations to match accordingly as requested

Reviewer #1: In this work, the Authors investigated by a systematic review and meta-analysis the effects and the safety of Roxadustat (ROX) in CKD patients not on dialysis therapy.

1. Some further meta-analyses were recently published (Wang et al., Ann Palliat Med 2021, 10:4736–46; Qie et al., Int Urol Nephrol 2021, 53:985–97; Zhang et al., Aging 2021, 13:17914–29; Liu et al., Front Med 2021, 8: 724456) and should be added to the article.

Reply: Thank you so much for your suggestions, we added the suggested articles to our discussion except Wang et al. as it discussed dialysis dependent patients not non-dialysis dependent patients. And our target population is non-dialysis-dependent patients.

2. The Authors report in the present work higher serious adverse events, hypertension, and deep venous thrombosis in ROX treated patients. By contrast, a recent meta-analysis (Liu, see above) showed that total adverse events were not significantly different between ROX and placebo as confirmed by trial sequential analysis, with increased risk of hyperkalemia events. A comment is welcomed.

Reply: Thank you so much for your comment, we added a comment on Liu et al. article in our discussion and commented about hyperkalemia as suggested.

3. Three phase 2 trials were included: may this decrease the level of evidence?

Reply: Thanks for your comment. We did a subgroup analysis to investigate the effect of the trial phase on the Hb level. We found it did not affect the result. Please refer to Fig S1 in S1 File.

4. How were managed studies reporting data for more than one ROX dose?

Reply: Thanks for your comment. The ROX doses were different between studies and we couldn’t do a subgroup analysis because multiple different doses were used in the included RCTs and there was no clear value that we can use to subgroup the ROX doses. We report that in the limitations.

5. The Authors report improved iron utilization parameters upon ROX treatment including decreases in ferritin and transferrin saturation and increases in TIBG and transferrin. These changes however also indicate a decrease in body’s iron storage and available iron, and suggest that patients were not receiving appropriate iron therapy for body iron requirement during ROX treatment. It appears that a stranded iron application is necessary in future studies to better characterize the effects of ROX on iron metabolism. Authors would comment on this issue.

Reply: Thank you so much for your input, we added this to our discussion

Reviewer #2: Abdelazeem and Colleagues, with this systematic review and meta-analysis, tried to extrapolate from the randomized studies available the efficacy and safety of Roxadustat on change in Hemoglobin and related parameters as well as on cardiovascular risk. The study is of interest and statistical analysis is overall of good quality. Moreover, this is an important topic from a clinical research perspective. However, I have several concerns to share with Authors:

1. Abstract: I would suggest Authors to change “efficacy and safety of ROX, especially on the cardiovascular system” into “efficacy and safety of ROX, especially on cardiovascular endpoints/risk”. The word system is too general. Moreover, please change “hat evaluated ROX NDD- CKD patients” with “hat evaluated ROX in NDD-CKD patients”;

Reply: Thank you So much for your comments. We edited that in the revision.

2. English language should be improved throughout the manuscript.

Reply: Thank you So much for your comments. We revised the whole manuscript grammatically and sent it for language editing by a professional.

3. Introduction is clearly written, and the aim is well depicted in my opinion.

Reply: Thank you so much for your comment.

4. Methodology is overall accurate in term of study selection and statistical tools used. However, I have a major concern namely the fact that studies were “intervention was ROX compared placebo or any other medications” were included in the metaanalysis. In this way Authors compared for example ROX with darbepoetin or ROX against no ESA. This can introduce a bias if the primary endpoint is the change in Hb levels.

Reply: Thank you so much for your comments. Only two studies used darbepoetin and the rest used placebo as the control group. We did a sensitivity analysis by omitting each study (Please refer to Table S2 in S1 File) and this didn’t affect the primary endpoint results. In addition that we did subgroup analysis ROX to placebo and ROX to darbepoetin alfa. We discussed the result in primary endpoint paragraph and the analysis can be seen in Fig S2 in S1 File.

5. Please also improve the quality of the figures. Most of them are not clearly interpretable in their current form.

Reply: Thank you so much for your comments, We tried to improve the quality. The figures currently are in high-quality TIF format with 300 dpi.

6. Some subgroup analyses/meta-analyses (for instance panel D of figure 4) included less then 5 studies and this confers low power to statistical findings. Please clarify this in the limitations if possible.

Reply: Thank you so much for your comments, we added this to our limitation as requested.

*********End of review********

Please address all correspondence concerning this manuscript to me at eltobgy.2@osu.edu

Thank you for your consideration of this manuscript.

Sincerely,

Mostafa Eltobgy, MD

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

17 Mar 2022

The efficacy and safety of roxadustat for the treatment of anemia in non-dialysis dependent chronic kidney disease patients: An updated systematic review and meta-analysis of randomized clinical trials

PONE-D-21-35500R1

Dear Dr. Eltobgy,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Gianpaolo Reboldi, MD, MSc, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The article has been improved in term of methodology and presentation of results. I will thus suggest acceptance

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Michele Provenzano

24 Mar 2022

PONE-D-21-35500R1

The efficacy and safety of roxadustat for the treatment of anemia in non-dialysis dependent chronic kidney disease patients: An updated systematic review and meta-analysis of randomized clinical trials

Dear Dr. Eltobgy:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

Prof Gianpaolo Reboldi

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