A Review of Critical Differences Among Loop Thiazide and Thiazide-like Diuretics
J Cell Mol Med. 2017 Nov; 21(11): 2634–2642.
Comparison of thiazide‐like diuretics versus thiazide‐type diuretics: a meta‐assay
Wenjing Liang
1 The Fundamental Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry building of Health, and The Country and Shandong Province Joint Cardinal Laboratory of Translational Cardiovascular Medicine, Qilu Hospital of Shandong University, Jinan, Shandong, China,
Hui Ma
ii Department of Pediatrics and Department of Cardiology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, Communist china,
Luxi Cao
3 Kidney Disease Center, Outset Affiliated Infirmary, Schoolhouse of Medicine, Zhejiang University, Hangzhou, Zhejiang, China,
Wenjiang Yan
ane The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Instruction and Chinese Ministry building of Health, and The State and Shandong Province Joint Fundamental Laboratory of Translational Cardiovascular Medicine, Qilu Infirmary of Shandong University, Jinan, Shandong, Mainland china,
Jingjing Yang
1 The Primal Laboratory of Cardiovascular Remodeling and Office Enquiry, Chinese Ministry building of Didactics and Chinese Ministry building of Health, and The State and Shandong Province Articulation Key Laboratory of Translational Cardiovascular Medicine, Qilu Hospital of Shandong University, Jinan, Shandong, China,
Received 2016 November 28; Accepted 2017 Mar 23.
Abstract
Thiazide diuretics are widely used for the management of hypertension. In contempo years, it has been actively debated that in that location is interchangeability of thiazide‐blazon diuretics hydrochlorothiazide and thiazide‐similar diuretics including indapamide and chlorthalidone for the treatment of hypertension. With the purpose of seeking out the all-time thiazide diuretic for clinicians, we summarized the existing evidence on the two types of drugs and conducted a meta‐analysis on their efficacy in lowering blood pressure level and effects on blood electrolyte, glucose and total cholesterol. Twelve trials were identified: v based on the comparing of indapamide versus hydrochlorothiazide and vii based on the chlorthalidone versus hydrochlorothiazide. In the meta‐analysis of blood pressure reduction, thiazide‐like diuretics seemed to further reduce systolic BP ([95% CI]; −5.59 [−5.69, −5.49]; P < 0.001) and diastolic BP ([95% CI]; −ane.98 [−3.29, −0.66]; P = 0.003). Meanwhile, in the analysis of side effects, the incidence of hypokalemia ([95% CI]; ane.58 [0.lxxx, 3.12]; P = 0.19), hyponatremia ([95% CI]; −0.xiv [−0.57, 0.xxx], P = 0.54), modify of claret glucose ([95% CI];0.thirteen [−0.16, 0.41], P = 0.39) and total cholesterol ([95% CI]; 0.13 [−0.16, 0.41], P = 0.39) showed that in that location is no statistical significant differences betwixt the 2 groups of drugs. In conclusion, using thiazide‐similar diuretics is superior to thiazide‐type diuretics in reducing claret pressure without increasing the incidence of hypokalemia, hyponatraemia and any change of claret glucose and serum total cholesterol.
Keywords: thiazide‐like diuretics, thiazide‐blazon diuretics, hypertension, hypokalemia, hyponatremia
Introduction
Thiazide diuretics were in one case the start effective oral antihypertensive agents with an acceptable side‐effect profile. For more than a half‐century, thiazide diuretics have been used for the management of hypertension 1. Despite structural variation among the heterogeneous grouping of agents including the thiazide‐type as well as thiazide‐similar diuretics, the term 'thiazide diuretic' incorporates all diuretics believed to accept a primary activeness in the distal tubule 1. The publication of 2014 'Bear witness‐Based Guideline for the Direction of High Claret Pressure level in Adults' from the Panel Members Appointed to the Eighth Joint National Committee (JNC 8) recommended the 'thiazide‐type diuretics' as first‐line therapy 2. In JNC 8, the recommendation is specific for diuretics, which includes thiazide‐like diuretics including indapamide and chlorthalidone, and information technology does not include loop or potassium‐sparing diuretics 3. Based on the evidence of some large‐calibration randomized clinical trials (RCTs), the condition of thiazide diuretics keeps playing a more vital function in treating hypertension.
Recently, the interchangeability of hydrochlorothiazide and chlorthalidone has been a matter of intense debate iv. Later on single oral doses, HCTZ accomplished peak concentrations in ≈two hr and had a one-half‐life of ≈6.5 to 9 hr. The one-half‐life of HCTZ would suggest that the drug should exist given twice daily. Compared to HCTZ, chlorthalidone has a longer one-half‐life, almost 42 60 minutes (range, 29–55 hr). At the same time, inspection of the bachelor studies suggests that 50 mg HCTZ is approximately equivalent to 25–37 mg chlorthalidone. In other words, it suggests that equivalent doses of chlorthalidone should generally exist 50–75% of typical HCTZ doses 5. Both HCTZ and chlorthalidone have demonstrated cardiovascular events take chances reduction in clinical trials. However, the largest trials, including, The Systolic Hypertension in the Elderly Program (SHEP) report 6, vii, elaborated that simply chlorthalidone tin significantly lower rates of stroke equally well as some other fatal or non‐fatal cardiovascular events. The Antihypertensive and Lipid Lowering Handling to Prevent Heart Attack (ALLHAT) study 8 that included over xxx,000 hypertensive patients compared chlorthalidone to alpha‐blockers, calcium channel blockers and ACE inhibitors. This randomized controlled trial demonstrated that at 12.5 and 25 mg/day dosages, chlorthalidone was beneficial in reducing new‐onset center failure compared with the other treatment used in the trial. Although at that place were potent show supporting the idea that chlorthalidone should be recommended every bit the initial handling of hypertension, a study comparing these two types of diuretics directly is missing.
Indapamide, a fellow member of thiazide‐like agents, presents some differences with others. In fact, besides its diuretic properties, indapamide presents some protective effects on vascular or organ harm in fauna and human investigations. In some clinical trials, information technology was effective in reducing left‐ventricular mass index in hypertensive patients and remarkably improving the renal function ix. Furthermore, in a recent big‐calibration RCT, employ of a sustained‐release indapamide‐based regimen as initial therapy led to relative reductions of 39%, 64% and 21% in the rates of fatal stroke, eye failure and death, respectively, in patients older than 80 years of age 10. Although much evidence demonstrated the effectiveness and advantages of indapamide, there is no big‐scale firsthand clinical trial and meta‐analysis to evaluate the BP lowering efficacy and safety betwixt indapamide and HCTZ.
To examine whether thiazide‐similar diuretics were superior over the thiazide‐blazon diuretics in lowering blood pressure without affecting the biochemical backdrop, we undertook a meta‐analysis of clinical trials with either HCTZ versus chlorthalidone or HCTZ versus indapamide as the contained comparative arms.
Methods
Data sources and search strategy
In our meta‐analysis, we adhered to the PRISMA guideline strictly. The search strategy was designed by the first author and reviewed by another two authors. We searched for evidence in PubMed (1948–2017/January) and Embase (1980–2017/January), Google Scholar, the ScienceDirect, the metaRegister of Controlled Trials and the Cochrane Primal Annals of Controlled Trials using the following terms: (indapamide OR chlorthalidone) AND (hydrochlorothiazide) AND(systolic blood pressure OR diastolic blood pressure)AND (cardiovascular events OR antihypertensive efficacy OR heart rates OR electrolyte disturbance OR hypokalemia OR hyponatremia OR serum cholesterol OR blood glucose OR hyperglycemia OR hypercholesteremia OR adverse effects). Moreover, references about the relevant reviews were likewise sought.
Inclusion and exclusion criteria
Trial inclusion criteria met the post-obit criteria: (i) randomized clinical trial or double‐blind controlled trial of thiazide‐like or thiazide‐blazon therapy in people with hypertension (SBP ≥ 140 mmHg or DBP ≥ xc mmHg); (two) patients were allocated to 2 monotherapy thiazide or combined with other kind of antihypertensive drugs in fixed‐dose arms; (3) duration of follow‐up ≥iv weeks; (iv) baseline washout and run‐in phase of medication ≥one calendar week; (5) measurements of ≥1 of the post-obit, systolic BP, diastolic BP, serum potassium, uric acid, serum cholesterol, glucose or heart rate. There were not yet plenty trials including thiazide‐like or thiazide‐type diuretics for meta‐analysis with the exception of hydrochlorothiazide, chlorthalidone and indapamide, and then only trials including the comparison betwixt hydrochlorothiazide and chlorthalidone or hydrochlorothiazide and indapamide were included. We excluded trials where subjects were predefined equally responders or non‐responders before the trial. If the study used the titration method in stride‐up protocols, we just extract the information using the initial doses. Studies using potassium supplementation were included, but stride‐down and drug withdrawal protocols were ineligible. Trials were besides ineligible if participants were <18 years old or had cirrhosis with ascites, type 1 or 2 diabetes, nephrotic syndrome, renal insufficiency, documented serum creatinine level >ane.5 times normal, history of ischaemic stroke, unstable angina or myocardial infarction within the by vi months, cardiac failure, secondary hypertension, dementia or other cognitive impairment, pregnancy or lactating women. If the written report condition permits, we used the blood force per unit area records via ambulatory claret pressure monitoring, if non, where resting BP measurements were bachelor for >ane time during a 24‐hr catamenia, the trough measurement, divers every bit 22–26 hr after dose, was used. When BP was recorded in multiple positions, sitting BP was used, unless variance data were only given for some other position, in which example that position was used.
Study characteristics and quality cess
The characteristics of studies xi, 12, xiii, 14, xv, 16, 17, xviii, nineteen, 20, 21, 22 used for the meta‐analysis were shown in Table 1. These studies were published from 1983 to 2016. A total number of 1580 patients from 12 trials were studied, in which 10 and 11 studies provided the data of systolic BP and diastolic reduction later treatment, respectively. In the thiazide‐like diuretics group, we integrated the indapamide and chlorthalidone together. Every bit there was lack of the head‐to‐head comparison trials betwixt chlorthalidone and HCTZ, nosotros constitute only three eligible trials used them in the monotherapy arm, while the remaining iv trials compared the chlorthalidone and HCTZ both combined with another drug. All of the included studies were published in English language. Score developed from the criteria of Jadad was utilized to appraise study quality 23, which had a possible range from naught to five, including double blinding, randomization and drop‐outs. It was defined as high quality if a study scored range from three points to five points.
Table 1
Study (author, year) | BP inclusion criteria | Quality score | Subjects (N) | Interventions | Duration of washout and run‐in (weeks) | Elapsing of handling (weeks) | Results measurement |
---|---|---|---|---|---|---|---|
Radevski et al. (2002) | Sitting DBP 95‐115 mmHg | three | 42 | 12.5 mg HCTZ vs. two.5 mg IND | 3 | 12 | 24‐hr ABPM |
David et al. (1999) | Sitting DBP 95–105 mmHg | 3 | 39 | 25 mg HCTZ vs. ii.5 mg IND | 4 | 24 | Supine BP |
GERARD et al. (1983) | Sitting DBP ≥ 95 mmHg | three | 24 | 50 mg HCTZ vs. 2.5 mg IND | vi | 12 | Recumbent BP |
A.Pareek et al. (2009) | SBP 140–179 mmHg or DBP xc–109 mmHg | three | 60 | 50 mg Metoprolol Twoscore + 6.25 mg CTD vs. 50 mg Metoprolol Xl + 12.5 mg HCTZ; | 1 | 8 | Seated BP; plasma potassium tape |
Leonetti et al. (2004) | Supine DBP 95–114 mmHg Supine SBP 161–209 mmHg | 4 | 354 | 25 mg HCTZ vs. one.5 mg Indapamide SR | 4 | 12 | Supine BP |
Kwon et al. (2012) | Never treated HTN(SBP ≥ 140 mmHg and/or DBP ≥ ninety mmHg) | iii | 28 | viii mg Candesartan + 25 mg HCTZ vs. 8 mg Candesartan + 12.5 mg CTD | iv | viii | Supine brachia BP |
Michael et al. (2006) | SBP 140–179 mmHg or DBP 90–109 mmHg | 4 | 24 | Firstly: 12.v mg CTD vs. 25 mg HCTZ; at week 4: force‐titrated to 25 mg CTD vs. 50 mg HCTZ | 4 | viii | 24‐60 minutes ABPM |
Pareek et al. (2009) | SBP 140–179 mmHg or DBP ninety–109 mmHg | 3 | 120 | 25 mg Losartan + six.25 mg CTD vs. 25 mg Losartan + 12.5 mg HCTZ | 2 | 4 | Office claret pressure measurement |
Senior et al. (1993) | DBP 95–120 mmHg | iv | xl | 25 mg HCTZ vs. two.5 mg IND | 2 | 24 | Diastolic blood pressure |
Siegel et al. (1992) | DBP 90–105 mmHg | 5 | 233 | 50 mg HCTZ vs. 50 mg CTD | 4 | 8 | 24‐hr Holter monitoring and laboratory tests |
Bakris et al. (2012) | Seated SBP 160–190 mmHg | four | 587 | twoscore mg AZL‐Thou/12.5 mg CTD vs. xl mg AZL‐Thou + 12.5 mg HCTZ | 4 | iv | 24‐60 minutes mean BP |
Anil Thousand. Pareek et al. (2016) | Office SBP betwixt 140 and 159 mmHg and DBP between 90 and 99 mmHg | 5 | 34 | 12.5 mg HCTZ vs. vi.25 mg CTD | ii | 12 | 24‐hour ambulatory blood pressure level monitoring |
Data extraction and statistical analysis
The primary end‐bespeak for our analysis was the systolic and/or diastolic BP reduction, the incidence of hypokalemia, hyponatremia and the change of serum full cholesterol and glucose throughout the different drug therapy. The changes of blood force per unit area, serum TG and glucose were computed every bit the difference in the BP values at the concluding follow‐upwards (or specific time‐betoken if multiple fourth dimension‐points were provided) compared to the baseline or initial measurement. Still, we analyse the incidence of hypokalemia using the dichotomous method. All data extracted from the 12 studies were recorded in Microsoft Excel before transfer to Review Manager version 5.0 program for assay. Continuous variables were conveyed in the course of the mean and standard difference (SD). Research results data whose ways and SDs were unavailable were kicked out of the meta‐assay. Heterogeneity was calculated using the I‐square statistic 24. I‐squared is the ratio of truthful heterogeneity to full variation in observed effect, and it volition not be impacted by the size of studies. All analyses were initially carried out using a fixed‐effects model. Notwithstanding, if heterogeneity beyond studies was observed, the analyses were carried out with a random‐effects model. The random‐furnishings model of inverse variance was used to summate the odds ratio (OR) with 95% conviction interval (95% CI).
Results
Characteristics of studies
A total of 2485 records were constitute after searching in PubMed, Embase, Google Scholar and other databases. As some literatures did not provide free full texts, or the inappropriate comparative methods, simply 45 full‐text articles were reviewed subsequently the first pick process (Fig. one). In the second pick process, we have filtered out 33 articles, which do not contain complete available data; lower than 3 when doing quality score assessment according to Jadad score; practise not have washout menstruation. Ultimately, twelve studies were maintained in our re‐analysis. The characteristic of included studies was described in this article (Table 1).
SBP reduction
In all of the included studies, 1307 patients from 10 trials provided information of systolic BP change from the baseline to the end‐signal of treatment period. Figure 2 shows the wood plot of different lowering systolic blood force per unit area efficacy betwixt thiazide‐like and thiazide diuretics. The final merged results show that thiazide‐similar diuretics (indapamide or chlorthalidone) will be significantly effective in lowering systolic BP(pooled effect size [95% CI]; −v.59 [−5.69, −five.49]; Heterogeneity: Chi² = 10.02, df = 9 (P = 0.35); I² = 10%) than thiazide‐type diuretics (hydrochlorothiazide).
DBP reduction
A meta‐analysis of diastolic BP reductions between the thiazide‐like and thiazide‐type diuretics besides showed a statistically meaningful result. As shown in Effigy 3, among 1347 subjects from 11 trials, compared to hydrochlorothiazide, indapamide or chlorthalidone, tin lower diastolic claret pressure level more finer (pooled outcome size [95% CI]; −1.98 [−3.29, −0.66]; Heterogeneity: Tau² = 2.90; Chi² = 66.81, df = 10 (P < 0.00001); I² = 85%). The heterogeneity exists.
Incidence of hypokalemia
Four trials provided data of incidence of hypokalemia happened during or later the treatment menstruation amongst 1050 subjects. It is shown in Figure iv, using thiazide‐similar diuretics indapamide or chlorthalidone, patients will have the same risks of hypokalemia with hydrochlorothiazide users (pooled result size [95% CI]; 1.58 [0.80, 3.12], P = 0.xvi; Heterogeneity: Tau² = 0.thirteen; Chi² = iv.ten, df = 3 (P = 0.25); I² = 27%).
Incidence of hyponatremia
As shown in Figure v, data from ii trials showed the same hazard of hyponatremia in users of thiazide‐like grouping and thiazide‐type group (pooled upshot size [95% CI]; −0.14 [−0.57, 0.thirty], P = 0.54; Heterogeneity: Chi² = 0.xiii, df = 1 (P = 0.71); I² = 0%).
Total cholesterol and glucose
4 trials included data from 550 subjects; as shown in Effigy 6, there were no statistically significant differences of total cholesterol between the two treatment groups (pooled consequence size [95% CI]; 0.11 [−0.02, 0.24], P = 0.xi; Heterogeneity: Tau² = 0.00; Chi² = 0.64, df = 3 (P = 0.89); I² = 0%). In the meta‐assay of change of serum glucose, we scanned seven trials included 804 subjects and the results also testify no meaning differences between thiazide‐like and thiazide‐type diuretics (pooled effect size [95% CI]; 0.13 [−0.16, 0.41], P = 0.39; Heterogeneity: Tau² = 0.07; Chi² = sixteen.35, df = 5 (P = 0.006); I² = 69%). The heterogeneity exists.
Heterogeneity and sensitivity analyses
We tin can believe that there is no heterogeneity exists in the analysis of systolic blood pressure reduction, incidence of potassium and total cholesterol because balmy heterogeneity might account for I2 is <30% of the variability in point estimates. However, in the meta‐analysis of diastolic blood pressure reduction and the alter of serum glucose, a notable heterogeneity is observed as Iii is more than 50% 23. To solve the problem, we have carried out the sensitivity analyses to exclude lower‐quality studies, merely there is no change of the results even when used another statistical model (data non shown).
Give-and-take
For meta‐analysis, we searched for published thiazide diuretic‐related studies that are 3 decades old. We extracted useful information from 12 included clinical trials. We reanalysed them and made a series of outcomes. The potent of thiazide‐type and thiazide‐like diuretics differed quite remarkably. We found out that thiazide‐like diuretics, indapamide and chlorthalidone, have an advantage in lowering both systolic and diastolic claret pressure without significantly increasing the risks of hypokalemia and hyponatremia, or making a meaning change of claret glucose and serum total cholesterol compared to the virtually prescribed thiazide‐type diuretics such as hydrochlorothiazide.
Thiazide diuretics are the second well-nigh mutual type of antihypertensive drug. Therefore, it continues to be widely used in the treatment of hypertension. Whereas in the past half‐century, there was few studies which compared the thiazide‐type and thiazide‐like diuretics direct in random trials. To provide more powerful evidence for clinical therapy, it is essential to make a comprehensive comparison betwixt the most common prescribed antihypertensive agents. In consideration of the side effects of thiazide diuretics, the major concern might be electrolyte disturbance, serum glucose and full cholesterol. Due to the longer one-half‐life of thiazide‐similar diuretics, risks of adverse events would be expected to increase, particularly the sodium and potassium homoeostasis disorders. Our analysis fabricated a comprehensive comparison of thiazide‐type and thiazide‐like diuretics to indicate that to attain the aforementioned level of BP reduction, thiazide‐similar diuretics were not worse than thiazide‐type diuretics in increasing risks of electrolyte disturbance.
Antihypertensive drugs are not simply able to lower blood pressure, just at that place are many extra benefits in the cardiovascular system, such equally anti‐inflammatory, anti‐atherosclerosis, ameliorate cardiac part and target organ protection. Several clinical studies had shown that low‐dose diuretics could exert target organ protection issue when compared to other antihypertensive 25, 26.
Our findings of this study should be gear up in context of previous meta‐analyses. In a dose‐stratified meta‐analysis and metaregression of 26 studies, Peterzan et al. 27 characterized the dose–response relationships for three commonly prescribed thiazide/thiazide‐similar diuretics, hydrochlorothiazide, chlorthalidone and bendroflumethiazide. He showed that chlorthalidone is more than effective than hydrochlorothiazide both for BP reduction and biochemical outcomes. He also provided us the recommended dose and dose–result human relationship of these diuretics. In another large‐calibration meta‐analysis published final twelvemonth 28, thiazide‐like diuretic could reduce risk of cardiovascular events and middle failure compared to thiazide‐type diuretics. In this meta‐analysis, the author used both placebo and other antihypertensive drugs as command arm, which would probably induce a biased baseline of the comparing. There was no detailed adverse result analysis in this article. To avert the divergence in baseline, we only included studies that used both thiazide‐like and thiazide‐type diuretics as different arm only in the same trial. Additionally, this is the start meta‐analysis, which highlighted the comparing of the major agin effects of these 2 diuretics.
Electrolyte disturbance might be a major business organisation for prescribing thiazide diuretic. January C. performed a case–command study 29, which plant an increase risk of hyponatremia in CTDN arm compared to HCTZ arm when given the equal dose, whereas in that location is no significantly increased incidence of hyponatremia in using CTDN compared with twice the dose of HCTZ per twenty-four hour period (CTDN 12.v mg/day vs. HCTZ 25 mg/day and CTDN 25 mg/twenty-four hours vs. HCTZ 50 mg/solar day). The dose‐titrate of CTDN and HCTZ was mentioned in Berkris et al. report eleven. Therefore, a lower dose of CTDN required to achieve the same BP reduction likewise equally cardiovascular outcomes might as well reduce the risk of hyponatremia. Likewise, in our study, we also performed the aforementioned results that using thiazide‐like diuretic would not increment the incidence of hyponatremia compared to HCTZ treatment.
Several limitations exist in our meta‐assay. Kickoff, in 2 of the included studies, the original pattern is a crossover study, only to insure statistical robustness of the data in the presence of a possible carryover upshot, nosotros decided that only the data from those who complete the starting time active handling period would be analysed. Secondly, we combined the indapamide and chlorthalidone together as 1 of the comparing arm without considering the variance between these two agents. On the other hand, we did not do the dose–response of the three drugs separately. This therefore ways that we cannot neglect the error from different doses in the aforementioned group. Thirdly, some of our included studies were published long ago with the differences in inclusion and exclusion criteria, BP measurement techniques and drug formulation. Although the score of these trials (as assessed by Jadad criteria) is more than than 3, the information from these studies volition contribute to heterogeneity between studies. As there is lack of head‐to‐head comparative trials between chlorthalidone and HCTZ, we used the information from a combination of them with beta‐blocker, ACEI or calcium channel blocker. The combination trials might brand it difficult to access the effects on blood pressure reduction efficacy, the adventure of incidence of hypokalemia and hyponatremia and the change of claret glucose and total cholesterol of thiazide diuretics, which would make the results of the meta‐analysis less compelling.
In summary, we performed a meta‐assay for the controversial clinical decision that whether thiazide‐type or thiazide‐like diuretics should be more prescribed as the initial antihypertensive therapy. Our assay obtained the different efficacy of BP reduction and biochemical outcomes when used the two kinds of drugs. It is recommended that further randomized controlled trials should exist done to compare these two types of drugs. The bachelor determination from our analysis suggests that thiazide‐type diuretic should be replaced by the thiazide‐like diuretic, which possess higher BP reduction efficacy and no more than risk of electrolyte disturbance and metabolic disorders.
Conflict of interests
No potential conflict of interests were disclosed.
Authors' contributions
W.J.50 designed and conducted the assay and wrote the manuscript. H.Chiliad and L.X.C collected and evaluated the data. J.J.Y and W.J.Y contribute to the design, analysis and revised the manuscript. J.J.Y is the guarantor of the work and takes the responsibleness and accuracy for the integrity of the results.
Acknowledgements
This work was supported by National Natural Science Foundation of Communist china (81500339, 81400195).
Correspondent Information
Wenjiang Yan, Electronic mail: moc.361@6067891jwy.
Jingjing Yang, E-mail: moc.liamg@demgnijgnijy.
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Articles from Periodical of Cellular and Molecular Medicine are provided here courtesy of Blackwell Publishing
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661252/
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