A special article following the relicence of aprotinin injection in Europe
David Royston a,*, Stefan De Hert b, Jan van der Linden c, Alexandre Ouattara d,e, Kai Zacharowski f
aCardiothoracic anaesthesia, critical care and pain, Royal Brompton and Harefield NHS foundation trust, Harefield hospital, Harefield, UK
bDepartment of anesthesiology, Ghent University, Ghent university hospital, De Pintelaan 185, 9000 Ghent, Belgium
cThoraxkliniken/Department of cardiothoracic surgery & anesthesiology, Karolinska institutet, Karolinska university hospital, 17176 Stockholm, Sweden
dDepartment of anaesthesia and critical care II, CHU de Bordeaux, 33600 Pessac, France
eINSERM, biology of cardiovascular diseases, U1034, university Bordeaux, 33600 Pessac, France
fKlinik fu¨r Ana¨sthesiologie, Intensivmedizin und Schmerztherapie, Universita¨tsklinikum Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
A R T I C L E I N F O
Available online 13 February 2017
Benefit-Risk analysis Relicense
Nordic Aprotinin Patient Registry (NAPaR) Conditions of use
Aprotinin injection (Trasylol1) has been relicensed for use during cardiac surgery throughout Canada and Europe. Nordic Group B.V. acquired the rights for aprotinin injection from Bayer Shering Pharma and will be wholly responsible for distributing aprotinin injection throughout Europe. This process has started in some countries and will continue throughout Europe over the next 2–3 years. Nordic had been supplying aprotinin on a ‘named patient basis’ in some countries such as the UK. This use was classified as unlicensed or off-label use and this will be changed to the licensed compound in due course.
The purpose of this contribution is to address four issues related to the relicense process:
ti first to suggest why aprotinin therapy is still required in modern cardiac surgical practice;
ti second to explain why regulators have allowed aprotinin to be relicensed based on its benefit/risk profile;
ti third, to explain the conditions regulators have imposed to allow this relicense process and in particular establishing the Nordic Aprotinin Patient Registry (NAPaR);
ti finally to point out some of the basics of the use of Aprotinin injection and to explain some of the potential difficulties
associated with its clinical use that regulators wanted to highlight.
1.First, why do I still need aprotinin in my practice? There is a body of opinion that suggests that:
ti blood products have become much safer;
ti patients having cardiac surgery with modern techniques are being transfused less;
ti tranexamic acid is just as effective as aprotinin (and is cheaper!).
So why bother with aprotinin?
1.1.Is blood transfusion safe?
There is no doubt that the risk of an unwanted transfer of a infective organism has been reduced over the years but it is equally obvious that giving blood transfusions is still not completely risk free. The UK haemovigilance group reports the Serious Hazards of Transfusion (SHOT) each year. In their 2015 report (available at http://www.shotuk.org), a total of 3288 reports were analysed.
* Corresponding author.
E-mail address: [email protected] (D. Royston).
There were 26 deaths in the report, 296 cases of acute transfusion reaction 280 instances when an incorrect blood component was
2352-5568/ Cti 2017 Socie´te´ ¸francaise d’anesthe´sie et de re´animation (Sfar). Published by Elsevier Masson SAS. All rights reserved.
transfused and 254 handling and storage errors. Red blood cell transfusion itself may be associated with increased morbidity caused by infectious, immunological, or pulmonary complications [1–3].
1.2.Are we administering less blood?
There is convincing data that transfusion rates are being reduced with time but blood transfusion has not been eliminated in our increasingly at-risk populations.
1.3.Why not give tranexamic acid to everyone as it is just the same as aprotinin?
A recent article from the cardiac surgery group in Montreal 
showed that transfusion of more than 4 units of red cells occurred in nearly a quarter of all of patients having heart surgery in their center between 2012 and 2015 despite the near 100% use of tranexamic acid. This led the authors to conclude there is still a need for an efficient blood-sparing agent.
There are also three other recent articles that have compared the outcome of patients during the period when aprotinin was available (up until November 2007) with the period after, when tranexamic acid was the only alternative. Firstly, a retrospective analysis from Toronto in Canada showed there was a lower incidence of massive hemorrhage, associated with a significant (50%) reduction in mortality in the very highest risk patients given aprotinin compared to tranexamic acid . Secondly, an analysis of data from Berlin showed a 50% lower mortality with aprotinin (7.5%) compared to tranexamic acid (16.2%) in patients having open cardiac chamber surgery . Finally, a complex analysis of the data from the Bristol cardiac center in the UK showed a 2.5-fold increase in mortality in their highest risk cardiac surgical patients since aprotinin was withdrawn . These studies reflect changes monitored before and after withdrawal of aprotinin and may thus have some bias and especially a time dependency bias. However the magnitude of the differences between treatments suggests that the clear signal of significant benefit of aprotinin in complex surgery will still be evident. Nonetheless this type of analysis highlights the paucity of good quality trials comparing aprotinin with lysine analogues in cardiac surgery practice.
2.Mechanism of action and efficacy are different for aprotinin and tranexamic acid
Aprotinin differs from tranexamic acid in its mechanism of action and efficacy profile . Tranexamic acid is a totally synthetic analogue of lysine that binds to the lysine-binding site of plasminogen to prevent it from being converted to plasmin. In this way, it acts as a pure antifibrinolytic. Studies investigating a dose-response relationship  demonstrated a plateau in the reduction in post-operative drains loss. This may relate to tranexamic acid saturating the plasminogen lysine-binding site. In turn this may account for why there is no agreed effective and safe dose. In contrast, aprotinin is an enzyme inhibitor that acts in the same manner as the body’s natural serine protease inhibitor against plasmin (alpha 2 plasmin inhibitor). Both of these enzymes have been shown to inhibit excess fibrinolytic bleeding associated with administration of tissue type plasminogen activator without preventing the physiological clot lysis associated with adminis- tration of this activator 
Given that aprotinin acts as an enzyme inhibitor it is not surprising that the reduction in bleeding and transfusion requirements following major surgery has a dose-response profile. This has been shown in randomized placebo controlled studies in cardiac  major orthopedic  and hepatic transplantation surgery .
Aprotinin also has a different efficacy profile in patients taking perioperative antiplatelet agents. In those patients receiving aspirin monotherapy prior to surgery there is a consistent effect to reduce transfusion with aprotinin and no benefit in the one study investigating tranexamic acid .
Aprotinin has significant benefits to reduce both red cell and platelet transfusions in those patients taking dual antiplatelet therapy with aspirin and a thienopyrridine operated with [13–15]
or without  cardiopulmonary bypass. The only study with tranexamic acid and thienopyrridine  is difficult to interpret from a European perspective. This multicenter study from China reported a rate of red cell transfusion of 76.6% in those patients taking neither antiplatelet therapy nor tranexamic acid and having first time revascularization surgery. These patients were reported
to receive 5.4 ti 4.9 (mean ti SD.) units of red cells and had a re- exploration rate of 6.5%. In addition 8% of patients required intra- aortic balloon counterpulsation and 96% received inotrope support. These results from a ‘control’ population are very much in excess of values found in European practice so any potential beneficial effect of administering tranexamic acid is difficult to judge.
3.Aprotinin has been shown to be dangerous so why have regulators allowed aprotinin to be relicensed?
A number of the discussions and arguments on this issue have been published previously [18–20].
The Bayer Company voluntarily suspended the marketing of aprotinin in November 2007 following the release of some preliminary data suggesting an increased mortality in aprotinin treated patients enrolled into a randomized study in Canada (Blood Conservation Using Antifibrinolytics in a Randomized Trial (BART), which was eventually published in May 2008 , along with the publication in the New England Journal of Medicine (NEJM) of three observational studies [22–24] questioning the safety of this agent. The data from the three observational studies had been reviewed by the Food and Drug Administration (FDA) in the United States of America in September of 2007 and found not to provide evidence to show a negative benefit/risk ratio for aprotinin in the licensed indication.
The reasons for this are complex and based on the methods of analysis of the data that were prospectively collected but retrospectively analyzed. To perform this analysis the authors of the studies used a statistical method called propensity analysis. This methodology attempts to take into account all of the other variables that may contribute to the outcome. These factors are termed confounders and have to be equally matched between groups before any conclusion of the significance of the outcome being associated with the treatment can be made. This matching can be done to produce pairs of data or mathematically using weighting systems based on regression analysis.
The initial publication suggesting adverse outcomes with aprotinin used data derived from a prospectively collected but retrospectively analyzed dataset associated with the McSPI consortium . The propensity score developed for this article included a number of ‘risk factors’ for adverse outcome, such as duration of education and annual income that advisors to the regulatory authority felt were inappropriate for inclusion in the model. The FDA statisticians reanalyzed the data using more conventional risk factors for adverse outcome to develop their propensity score. This independent analysis found no significant association between aprotinin use and adverse outcomes apart from need for new dialysis. This aspect was explained by two factors. Firstly, centers in Germany contributed over 70% of all patients who received aprotinin to the dataset. Secondly, a subsequent publication using the same McSPI dataset showed
there was a very high incidence of new dialysis in patients operated on in these German centers . In fact this latter manuscript showed the incidence of new dialysis/hemofiltration was higher than the incidence of a predefined significant increase in plasma creatinine implying this intervention was used for non- renal reasons. When geography was included in the propensity model there was no evidence in the McSPI data for any adverse effect of aprotinin therapy on mortality, renal function, myocardial infarction or stroke.
The second manuscript was from an analysis of a very large patient population . The original data were derived from an administrative database used by the Premier Hospitals group in the United States to ensure fiscal stability. In addition to an increased mortality signal in the aprotinin treated patients this analysis also showed a highly significant reduction in mortality associated with having hypertension, currently smoking, having cancer, having had a recent myocardial infarction or stroke and being older. The regulatory authority felt that these anomalies probably reflect the lack of clinically relevant confounders in the dataset which precluded them making any inferences to suggest risk or otherwise of aprotinin therapy based on this analysis.
The final conclusions from another retrospective review of a prospectively recorded dataset  were deemed not to be appropriate for inclusion in a benefit/risk analysis, as the various confounders were not matched between the patient groups. Supplementary data containing a ‘matched pair’ analysis was available via the NEJM website  and this showed no mortality signal at 30 days or 1 year. However even this analysis failed to match the groups for age, red cell transfusion and year of surgery.
The BART study  had the benefit of being a blinded randomized controlled trial. The conclusion reached by the authors was that aprotinin was no more effective than tranexamic acid at preventing the primary end-point of massive bleeding and was associated with an increased mortality. However, some criticisms about the initial conclusion led the regulatory authority in Canada to call together an independent expert advisory panel (EAP), which met in December 2008.
They concluded, there were a number of limitations to the BART analysis [27,28]. The first of the main two identified was the exclusion of 137 patients from the analysis after randomization. This cohort included a number of dead patients, none of whom received aprotinin. When these patient data were included in the analysis the mortality signal became non-significant. The panel concluded that while there were numerically more deaths in the aprotinin-treated patients this could have occurred by chance.
In addition the balance of the deaths was highly skewed. Using the mortality in the first 5 days after surgery as more likely to reflect a drug related problem, the increased numerical mortality in patients allocated to receive aprotinin appeared to be focused on only 5 centers. It is difficult to explain why a drug that may be causally related to mortality only does this in about a quarter of centers. Their analysis did however show that anticoagulation control with heparin might have been suboptimal in these 5 centers due to the effect of aprotinin on tests of the intrinsic coagulation system [27,28].
Further analysis by Health Canada failed to show any relationship between risk stratification, hemodynamic variables and mortality (which was mainly cardiovascular in origin in all sites).
The second point Health Canada raised related to the reclassification of end points. The BART study included two planned interim analyses each after recruitment of a third the planned number of patients. The second of these was in January 2007 and included data from 1896 randomised patients . In this supplementary data , the incidence of the primary outcome (drains loss of > 1500 mL in the first 8 postoperative
hours, transfusion of 10 or more units of packed red cells, re- exploration due to bleeding or death due to hemorrhage in the 24 hours after protamine administration (changed to 30 days for the final publication) occurred in 7.2% (n = 50) of aprotinin treated patients. Following the addition of the 435 patients recruited between January 2007 and October 2007 the primary outcome end-points now occurred in 74 aprotinin allocated patients. This means that, assuming randomisation was equal, the primary outcome end point was observed in 24 of 145 patients or over 17% of aprotinin patients in the last cohort recruited. The EAP concluded about this last group of patients:
‘‘More worrisome was an unusually large number of reclassi- fications of outcomes from the originally reported data, with a large (approximately 75%) change rate in primary outcome (massive postoperative bleeding). Reclassifications were in opposite directions for aprotinin versus tranexamic acid and aminocaproic acid, favouring the latter, and these changes increased with the duration of the study.’’
The independent expert panel and Health Canada stated that these changes in a direction opposed to the interim analyses were never satisfactorily explained to them .
Some 5 years after this judgment the BART investigators wrote an article  intending to justify their claim for increased mortality with aprotinin. The article also questioned the indepen- dent experts and regulators that helped Health Canada and the European regulators come to their conclusion to relicense aprotinin. The analysis is complex and does not include raw data by multiple risk ratios. This makes it difficult to interpret when the totality of the BART data is analyzed. In particular, the BART drug safety monitoring board explained why they recommended stopping the study in the published supplementary data . At the time of this recommendation (October 2007) there had been 49 deaths in the aprotinin allocated patients with 30 in the tranexamic acid group and 31 in those allocated to epsilon aminocaproic acid. These are the same numbers as published in the main BART manuscript . However, at the time of the recommendation data from only 2163 patients had been analyzed and the comparison between agents for mortality had not reached conventional levels of statistical significance. It is difficult to explain why there were no more deaths in the additional 168 patients included in the final manuscript (305 patients if Health Canada analysis is appropriate) and this inclusion made the mortality difference highly significant.
Finally in Table 4 of the supplementary BART data  red cell transfusion was reported in 419 of 780 patients (53.7%) allocated to the aprotinin group and 506 of 770 (65.7%) of those allocated to tranexamic acid (P < 0.001) showing a highly significant advan- tage of aprotinin over tranexamic acid on transfusion burden in this group of patients despite the conclusion of the main article that there was no efficacy difference.
Given that independent analysis and review of the data on aprotinin therapy showed no conclusive safety signal, regulators in Canada agreed to relicense aprotinin for use during cardiac surgery.
The European Medicine Agency, together with their indepen- dent expert panel also revisited the totality of the aprotinin data and recommended the license be restored for aprotinin use in Europe. Despite convincing literature for an efficacy benefit in non- cardiac surgery [10,11] and a powerful anti-inflammatory effect of aprotinin during cardiac surgery , the totality of the data for the safety of aprotinin was largely derived from randomized placebo controlled studies designed to attain regulatory approval world- wide which were performed mainly in patients having myocardial
revascularization. Primarily for this reason the market authoriza- tion indication for use is currently:
‘‘To reduce blood loss and blood transfusion in adult patients who are at high risk of major blood loss undergoing isolated coronary artery bypass graft surgery using cardiopulmonary
Despite evidence for a benefit of weight related dosages to reduce intra-patient variability in plasma aprotinin concentrations [35,36] the regulatory authority in Europe has approved two fixed dosage regimen as shown below.
Regimen A (full-dose)
Regimen B (half-dose)
Test dose 10,000 KIU 10,000 KIU
4.What conditions have regulators in Europe imposed to enable the relicense process?
The regulators acknowledged that there would be use of aprotinin away from this indication and thus requested to have a registry of use of aprotinin (NAPaR) designed to establish not only the pattern of use of aprotinin throughout Europe but also if there are any specific safety issues in the population not classified in the current indication.
The regulatory authorities highlighted three specific issues that came out of their various reviews of the data that are now emphasized in the NAPaR documentation.
Initial or loading dose
Pump prime dose
Continuous infusion. Given until skin closure
2ti 106 KIU 280 mg
2 ti 106 KIU 280 mg
0.5 ti 106 KIU/h 70 mg/h
1 ti 106 KIU 140 mg
1 ti 106 KIU 140 mg
0.25 ti 106 KIU/h 35 mg/h
Firstly, Health Canada highlighted that the way heparin was used for anti-coagulation during cardiopulmonary bypass in the BART study was inconsistent [27,28]. Because the agents used to monitor the integrity of the intrinsic pathway are known to be influenced by aprotinin, this could potentially lead to insufficient anticoagulation.
Secondly, the regulatory authorities noted that that aprotinin therapy was associated with a small, transient but statistically significant plasma creatinine rise of ti 44 mmol/L in the days after surgery. Typically this rise occurred in about 8% of treated patients, compared to 5% in the placebo group and lasted for 9 days, compared to 5 days in placebo. Both these differences were statistically significant with P < 0.05. The mechanism is thought to be the same as found with basic chemicals  such as aminoglycosides and contrast media and is due to the proximal tubule reuptake mechanism being overloaded by the aprotinin being recycled from the glomerular filtrate. There is no evidence that use of aprotinin therapy is associated with an increased risk of renal failure or need for renal support therapy. The NAPaR thus contains fields of documentation concerning the timing of angiography and use of aminoglycosides together with data on plasma creatinine changes.
The final safety issue is of hypersensitivity reaction especially after a second exposure [33,34]. This is most likely in the 6 months following the first exposure and has been reported as presenting from a minor rash up to a lethal reaction. The NAPaR contains documentation about exposure to fibrin glue or to performing an antibody assay for aprotinin (which is not available commercially as a diagnostic tool for humans). The recommendation from the regulators is that aprotinin is not administered within 12 months of the first exposure.
Finally, the results from analysis of NAPaR will be used by regulators to guide changes in indications or the need for more formal randomized studies in specific patient populations.
5.Is there a simple instruction set for the clinical use of aprotinin?
Aprotinin is a polypeptide containing 58 amino acid residues. It is presently calibrated in Kallikrein Inhibiting Units (KIU). The commercial agent for human use is presented in 50 mL glass vials at a concentration of 10,000 KIU/mL in sterile 0.9% saline. Each vial therefore contains 500,000 KIU, which converts to 70 mg of polypeptide.
Although there are data to suggest that the elimination half-life of aprotinin is almost doubled in patients with an estimated creatinine clearance of < 25 mL/min  there was no greater increase in postoperative plasma creatinine than in patients with normal renal function. This study used an equivalent of the half- dose regimen and the European regulatory authorities have not advocated an alternate dosing scheme for patients with severely impaired renal function.
If hemofiltration is used during bypass then aprotinin will be freely filtered and removed from the circulation. This is because the commercial filters have a molecular cut off of about 14,000 Dalton and aprotinin has a molecular weight of 6500 Dalton. This will not affect the plasma concentration unless the filtered volume is replaced during this period.
ti Prior to surgery the assumption made is that a baseline value for anticoagulation control and arterial blood gas analysis has been performed.
ti Administer a test dose of 1 mL of solution taken from a 50 mL a vial after establishing arterial and wide bore venous vascular access and following induction of anaesthesia, endotracheal intubation and establishing ventilation.
ti It is not only common sense but also recommended that adrenaline is available to give an immediate dose in case of a hypersensitivity reaction.
ti After a few minutes infuse the initial/loading bolus over a 15 to 20 minute period. Rapid infusion has been associated with a fall in arterial blood pressure of about 20 mmHg in about 30% of patients. This may be significant in patients with tight left main stem or ostial lesions or aortic stenosis.
ti Aprotinin does not cause thrombophlebitis and can be delivered through a peripheral vein while central venous access is established. Preloading the loading dose into a ‘Buretrol’ ‘Soluset’ or similar infusion system facilitates this.
ti Only give the perfusionist the vials for the pump-prime dose chosen after this stage has passed.
When central venous access has been established the continu- ous infusion part of the dosing can be administered by syringe drive through one of the lumens. The current summary of product characteristics approved by the European regulators restates comments in prior documentation regarding not infusing aprotinin with another drug and especially heparin. This advice does not apply to the current formulation of aprotinin, which does not
neutralize heparin  but is related to a period when aprotinin contained an alcohol as a preservative that precipitated heparin.
Aprotinin is not a heparin-sparing agent so the initial bolus of unfractionated heparin should be as usually given in any specific center, typically 300–350 IU/kg. In patients undergoing cardiopul- monary bypass with aprotinin therapy, one of the following methods is recommended to maintain adequate anticoagulation:
5.1.Activated Clotting Time (ACT)
An ACT is not a standardized coagulation test, and different formulations of the assay are affected differently by the presence of aprotinin. Variable dilution effects further influence the test and the temperature experienced during cardiopulmonary bypass. It has been observed that kaolin-based ACTs are not increased to the same degree by aprotinin, as are diatomaceous earth-based (celite) ACTs. While protocols vary, a minimal celite ACT of 750 seconds or kaolin ACT of 480 seconds, independent of the effects of haemodilution and hypothermia, is recommended in the presence of aprotinin. For centers using the Hemochron Signature or Elite system the ACT+ cartridge contains silica, kaolin and phospholipid and data from the manufacturer suggest the result is unaffected by standard doses of aprotinin . In this case a value of 400 seconds may be appropriate for adequate heparinisation. However, this Hemochron instrument can also be used with an ACT-LR cartridge that contains only diatomaceous earth so the result should be treated as if using a celite cartridge.
5.2.Fixed Heparin Dosing
A standard loading dose of heparin, administered prior to cannulation of the heart, plus the quantity of heparin added to the prime volume of the cardiopulmonary bypass circuit, should total at least 350 IU/kg. Additional heparin should be administered in a fixed-dose regimen based on patient weight and duration of cardiopulmonary bypass.
5.3.Determination of Heparin Levels
Protamine titration, a method that is not affected by aprotinin, can be used to measure heparin levels. A heparin dose response, assessed by protamine titration, should be performed prior to administration of aprotinin to determine the heparin-loading dose. Additional heparin should be administered on the basis of heparin levels measured by protamine titration. Heparin levels during bypass should not be allowed to drop below 2.7 U/mL or below the level indicated by heparin dose-response testing performed prior to administration of aprotinin.
In aprotinin treated patients, the neutralization of heparin by protamine after discontinuation of cardiopulmonary bypass should either be based on a fixed dose (typically 2 mg/kg) a fixed ratio to the amount of heparin applied (typically 1 mg protamine for each 100 IU heparin) or be controlled by a protamine titration method.
Aprotinin use is associated with prolongation of all of the current commercial methods of measuring activated partial thromboplastin time (APTT). This test will therefore be prolonged in the immediate post-operative period and should not be used to guide therapy with coagulation factors and especially frozen plasma. The half-life of aprotinin in the circulation is about 150 minutes and with adequate urine output the APTT should not be significantly affected after about 6–12 hours.
It is over ten years since aprotinin was voluntarily withdrawn from European markets. The relicense has raised some unique
issues for not only regulators and the marketing authorization holder but also clinicians. Cardiac surgical practice and the patient risk profile have changed noticeably over the past 10 years and some clinicians may think they have established ways to combat the differing risk so will not consider a return to use of aprotinin . For those that do, the NAPaR will provide data on the pattern of use of aprotinin in Europe in the current age and more importantly should provide an early warning of any safety concerns that may arise.
Disclosure of interest
The authors of this work are all members of the independent drug safety monitoring committee (DSMC) (Chair Professor De Hert) for the Nordic Aprotinin Patient Registry (NAPaR) established by Nordic Pharma BV as a requirement of the European regulatory agency.
Professor De Hert declares that he have no competing interest. Professor van der Linden declares that he have no competing
Professor Ouattara has received honoraria as consultant to LFB, The Abbvie Pharmaceutical Company, The Medicines Company and Nordic Pharma.
Dr Royston received honoraria as a consultant to Bayer Schering Pharma during their meetings with regulators in September 2006, October 2007 and October 2010. He has also received honoraria for presentations and consultancy from Nordic Pharma BV.
Professor Zacharowski reports receiving honoraria for pre- sentations and consultancy together with financial support from a number of companies to support research and educational activity within his Department in Frankfurt. Among these companies are Abbott GmbH, AbbVie Deutschland GmbH, Astellas Pharma GmbH, AstraZeneca GmbH, Aventis Pharma GmbH, B. Braun, Melsungen AG, Baxter Deutschland GmbH, Bristol-Myers Squibb GmbH,CSL Behring GmbH, Dra¨ger Medical GmbH,Fresenius Kabi GmbH,Gam- bro Hospal GmbH, GlaxoSmithKline GmbH,Gru¨nenthal GmbH, Janssen-Cilag GmbH, Masimo, Novartis Pharma GmbH,Novo Nordisk Pharma GmbH, Pfizer Pharma GmbH, Siemens Healthcare, Vifor Pharma GmbH.
Dr Royston accepts on behalf of his co-authors that the work described has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors and if accepted, it will not be published elsewhere including electronically in the same form, in English or in any other language, without the written consent of the copyright-holder.
All authors have made significant contributions to writing and subsequent revisions of the current manuscript.
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