Intermittent hemodialysis is superior to continuous veno-venous hemodialysis/hemodiafiltration to eliminate methanol and formate during treatment for methanol poisoning

Abstract

During an outbreak of methanol poisonings in the Czech Republic in 2012, we were able to study methanol and formate elimination half-lives during intermittent hemodialysis (IHD) and continuous veno-venous hemodialysis/hemodiafiltration (CVVHD/HDF) and the relative impact of dialysate and blood flow rates on elimination. Data were obtained from 11 IHD and 13 CVVHD/HDF patients. Serum methanol and formate concentrations were measured by gas chromatography and an enzymatic method. The groups were relatively comparable, but the CVVHD/HDF group was significantly more acidotic (mean pH 6.9 vs. 7.1 IHD). The mean elimination half-life of methanol was 3.7 and formate 1.6h with IHD, versus 8.1 and 3.6h, respectively, with CVVHD/HDF (both significant). The 54% greater reduction in methanol and 56% reduction in formate elimination half-life during IHD resulted from the higher blood and dialysate flow rates. Increased blood and dialysate flow on the CVVHD/HDF also increased elimination significantly. Thus, IHD is superior to CVVHD/HDF for more rapid methanol and formate elimination, and if CVVHD/HDF is the only treatment available then elimination is greater with greater blood and dialysate flow rates. During an outbreak of methanol poisonings in the Czech Republic in 2012, we were able to study methanol and formate elimination half-lives during intermittent hemodialysis (IHD) and continuous veno-venous hemodialysis/hemodiafiltration (CVVHD/HDF) and the relative impact of dialysate and blood flow rates on elimination. Data were obtained from 11 IHD and 13 CVVHD/HDF patients. Serum methanol and formate concentrations were measured by gas chromatography and an enzymatic method. The groups were relatively comparable, but the CVVHD/HDF group was significantly more acidotic (mean pH 6.9 vs. 7.1 IHD). The mean elimination half-life of methanol was 3.7 and formate 1.6h with IHD, versus 8.1 and 3.6h, respectively, with CVVHD/HDF (both significant). The 54% greater reduction in methanol and 56% reduction in formate elimination half-life during IHD resulted from the higher blood and dialysate flow rates. Increased blood and dialysate flow on the CVVHD/HDF also increased elimination significantly. Thus, IHD is superior to CVVHD/HDF for more rapid methanol and formate elimination, and if CVVHD/HDF is the only treatment available then elimination is greater with greater blood and dialysate flow rates. Methanol poisoning is a medical emergency where rapid elimination of the toxin and its metabolite is crucial for recovery. This is because the accumulation of the toxic metabolite formic acid/formate is cytotoxic through the inhibition of mitochondrial respiration.1.Liesivuori J. Savolainen H. Methanol and formic-acid toxicity - biochemical-mechanisms.Pharmacol Toxicol. 1991; 69: 157-163Crossref PubMed Scopus (284) Google Scholar, 2.Hovda K.E. Hunderi O.H. Rudberg N. et al.Anion and osmolal gaps in the diagnosis of methanol poisoning: clinical study in 28 patients.Intensive Care Med. 2004; 30: 1842-1846Crossref PubMed Scopus (94) Google Scholar, 3.McMartin K.E. Ambre J.J. Tephly T.R. Methanol poisoning in human-subjects - role for formic-acid accumulation in the metabolic-acidosis.Am J Med. 1980; 68: 414-418Abstract Full Text PDF PubMed Scopus (230) Google Scholar, 4.Hovda K.E. Andersson K.S. Urdal P. et al.Methanol and formate kinetics during treatment with fomepizole.Clin Toxicol. 2005; 43: 221-227Crossref PubMed Scopus (49) Google Scholar In addition, the accumulation of formic acid results in a metabolic acidosis, visual impairment, and damage of the basal ganglia, especially when its concentration rises to 9–11mmol/l.5.Jacobsen D. McMartin K.E. Methanol and ethylene glycol poisonings.Mechanism of toxicity, clinical course, diagnosis and treatment. Med Toxicol. 1986; 1: 309-334Google Scholar, 6.Sejersted O.M. Jacobsen D. Ovrebo S. et al.Formate concentrations in plasma from patients poisoned with methanol.Acta Med Scand. 1983; 213: 105-110Crossref PubMed Scopus (108) Google Scholar, 7.Osterloh J.D. Pond S.M. Grady S. et al.Serum formate concentrations in methanol intoxication as a criterion for hemodialysis.Ann Intern Med. 1986; 104: 200-203Crossref PubMed Scopus (84) Google Scholar, 8.Salek T. Humpolicek P. Ponizil P. Metabolic disorders due to methanol poisoning.Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013https://doi.org/10.5507/bp.2013.5074Crossref Scopus (8) Google Scholar The role of hemodialysis in methanol poisoning is well established.9.Jacobsen D. Ovrebo S. Sejersted O.M. Toxicokinetics of formate during hemodialysis.Acta Med Scand. 1983; 214: 409-412Crossref PubMed Scopus (35) Google Scholar,10.Barceloux D.G. Bond G.R. Krenzelok E.P. et al.American Academy of Clinical Toxicology practice guidelines on the treatment of methanol poisoning.J Toxicol-Clin Toxicol. 2002; 40: 415-446Crossref PubMed Scopus (504) Google Scholar Hemodialysis eliminates both methanol and formate, and helps to correct metabolic acidosis. Both intermittent and continuous modalities of hemodialysis are commonly used in the treatment of poisonings; however, comparative studies are scarce for methanol kinetics and nonexistent for formate kinetics. Given the fact that ~80% of all dialysis sessions in 2006 were performed in the developed world,11.Aviles-Gomez R. Luquin-Arellano V.H. Garcia-Garcia G. et al.Is renal replacement therapy for all possible in developing countries?.Ethn Dis. 2006; 16: 70-72Google Scholar whereas the majority of methanol poisoning outbreaks occur in underdeveloped countries where resources are scarce; a thorough evaluation of the efficacy and limitations of the various modalities of treatment should be carried out. In addition, challenges regarding the availability of antidotes in some countries (for example, the general lack of fomepizole in the developing world, and cultural limitations regarding the medicinal use of ethanol in some Islamic countries) further emphasize the importance of extracorporeal elimination techniques in the treatment of patients with methanol toxicity. In addition to the availability of different dialysis equipments, the choice of modality may be governed by the patient’s clinical condition, especially with regard to the circulatory status. The risk of performing a dialysis session with a high dialysate and blood flow rate in a patient with a very low blood pressure has to be weighed against the benefits of the increased elimination rate of the toxic substances and correction of the acidosis. We therefore also wanted to study the relative impact of the blood and dialysate flow on the elimination of both methanol and formate to see whether this could add to the recommendations for the treatment. The above questions were addressed in a prospective observational study on methanol and formate elimination in 24 methanol-poisoned patients treated with antidotes and two different methods of enhanced elimination in order to compare the elimination half-lives of methanol and formate. All patients were treated during the recent outbreak of mass methanol poisoning in the Czech Republic in 2012, where 121 cases of poisoning were admitted and a total of 41 patients died (of whom 21 died in hospital). The epidemiological description of the outbreak is presented elsewhere.12.Zakharov S. Pelclova D. Navratil T. Methanol outbreak in the Czech Republic 2012: epidemiology, clinical features and outcomes. In preparation. 2013Google Scholar The clinical features, laboratory data, and patient outcomes are presented in Table 1. Among the 24 patients, 11 were treated with intermittent hemodialysis (IHD), whereas 13 were treated with a continuous modality (eight with continuous veno-venous hemodialysis (CVVHD) and five with continuous veno-venous hemodiafiltration (CVVHDF)). In two patients (patients nos. 8 and 11, Table 1), ethanol infusion was started before the initial blood sample was drawn, explaining why they were acidotic in spite of their serum ethanol concentration.Table 1Laboratory and clinical data of 11 patients treated with IHD and 13 patients treated with CVVHD/HDF on admissionSex/age (years)pHBase deficit (mmol/l)pCO2 (kPa)HCO3− (mmol/l)S-Methanol (mmol/l)S-Formate (mmol/l)Lactate (mmol/l)S-Ethanol (mmol/l)Osmolal gap (mOsm per kg H2O)Anion gap (mmol/l)Antidote givenFolate substitutionClinical featuresMean arterial pressure (mm Hg), pulse rate (min)Glasgow coma scaleSequelae1M306.7138ND4.3872011.40.012854.8FoFAVD.D.C79, 1023VS2M367.19202.98.624172.80.04228.1EFLAVD.D.GI117, 10015—3F527.20134.613.7166151.50.017325.3EFAGI100, 7515—4M587.17213.66.81316ND0.02828.8EFLAVD. HA114, 8015—5M797.32113.513.41054.40.01819.2ENoVD.GI107, 8015DiedaThe patient died in the hospital from infectious complications 1 month after the methanol poisoning.6F436.9327ND7.656178.00.0NDNDFoFAVD.GI.D.CP.C84, 1003Died7M366.98271.52.55244.00.013241.9FoFLAVD.GI.CP.HA127, 9912—8F587.14222.56.038112.536bBolus of ethyl alcohol was administered before blood sampling.Bold values indicate statistically significant P-values, P<0.05 (5%).5234.6ENoGI.D.F.IE127, 8513—9F627.17183.59.330162.00.03030.5ENoVD.GI.F.D113, 11015—10F587.11213.47.8105182.13.09123.6FoNoVD.GI117, 7015—11F627.08242.75.755170.911bBolus of ethyl alcohol was administered before blood sampling.Bold values indicate statistically significant P-values, P<0.05 continuous veno-venous IHD, intermittent the of a the the of a the visual visual The patient died in the hospital from infectious complications 1 month after the methanol of ethyl alcohol was administered before blood sampling.Bold values indicate statistically significant P-values, P<0.05 in a continuous veno-venous IHD, intermittent the of a the the of a the visual visual The two groups of patients treated with different of enhanced elimination were by to and of patients. The mean to diagnosis and treatment after the toxic alcohol was in the CVVHD/HDF group and in the IHD All data were with two serum ethanol in both groups of patients, and serum methanol in the CVVHD/HDF were the two groups in data with to serum formate, and osmolal gap The patients treated with CVVHD/HDF were more acidotic treated with IHD (mean pH vs. with higher (both Data on formate and methanol elimination in 11 patients treated with IHD and 13 patients treated with CVVHD/HDF are presented in and and methanol kinetics in 11 patients treated on during of data after flow IHD, intermittent the of a the the of a the used in all in a Table and methanol kinetics in the patients treated on S. Pelclova D. Navratil T. Methanol outbreak in the Czech Republic 2012: epidemiology, clinical features and outcomes. In preparation. 2013Google Scholar, D. H. et on methanol Med Scand. PubMed Scopus Google Scholar, D. McMartin K.E. for methanol and ethylene glycol poisoning.J Toxicol. PubMed Scopus Google Scholar, S. techniques in the treatment of Full Text PDF PubMed Scopus Google Scholar, et of methanol intoxication with hemodialysis an J Dis. 30: Full Text PDF PubMed Scopus Google Scholar, J. et use of an and dialysate to serum ethanol during for methanol PubMed Scopus Google Scholar, et hemodialysis for the treatment of patients with methanol J 2005; Scholar, R. R. et of hemodialysis with for the treatment of alcohol Scholar and G. in the poisoned PubMed Scopus Google Scholar, P. P. kinetics in methanol Toxicol. 2005; PubMed Scopus Google Scholar, McMartin et al.Formate kinetics in methanol poisoning.J Toxicol. 2002; 40: PubMed Scopus Google Scholar, D. R. et al.Methanol and formate kinetics in methanol PubMed Scopus Google Scholar, J. McMartin S. et for the treatment of methanol J Med. PubMed Scopus Google during of data of of during continuous veno-venous flow the of a the the of a the used in all in a flow IHD, intermittent the of a the the of a the used in all during continuous veno-venous flow the of a the the of a the used in all The mean methanol elimination half-life in study was for IHD and for The mean formate elimination half-life was during IHD and during The elimination half-lives of both formate and methanol on IHD were significantly on CVVHD/HDF (both of the of the elimination half-lives of methanol and formate were when the blood flow rates were higher (both half-life of formate versus blood flow rate In the CVVHD/HDF the elimination half-lives were when the dialysate flow rate was higher and when the was (both In IHD, only dialysate flow rate and only two of were used a significant was the and the elimination half-life of methanol half-life of methanol versus significant was the elimination half-lives of methanol and formate and the serum concentrations of formate, and a significant was the elimination half-life of formate and arterial blood the half-life of formate was in more acidotic patients significant of methanol and formate elimination half-lives were with the with and with the the the patients with visual and of methanol poisoning significantly elimination half-lives of formate The in and with IHD and CVVHD/HDF groups were The results of the of the elimination half-lives of formate and methanol on all 24 cases are in and in the of the formate elimination half-life The significant was the blood flow The dialysate flow rate was on the CVVHD/HDF cases however, was significantly to the The was as with explaining why is as the in the in spite of the both the methanol and the formate elimination The of coma mean arterial and pulse The and the gap The two the of the of formate elimination half-life flow flow of a the of of a the of and dialysate coma mean arterial pressure and pulse deficit and the gap in a Table of methanol elimination half-life flow of a the of of a the of and the gap in a of a the of of a the of and dialysate coma mean arterial pressure and pulse deficit and the gap of a the of of a the of and the gap the methanol elimination the of of the In this the dialysate flow rate and the blood flow rate were the the of whereas to this to the of the Thus, this the in of two of The for methanol was to the the clinical of the patient and more to the of hemodialysis as with the for formate. The was the and the the of metabolic acidosis, and the of poisoning in this Methanol poisoning is of the in clinical where dialysis has an D.G. Bond G.R. Krenzelok E.P. et al.American Academy of Clinical Toxicology practice guidelines on the treatment of methanol poisoning.J Toxicol-Clin Toxicol. 2002; 40: 415-446Crossref PubMed Scopus (504) Google D. H. et on methanol Med Scand. PubMed Scopus Google Scholar Both methanol and formate are and with a of for D. McMartin K.E. for methanol and ethylene glycol poisoning.J Toxicol. PubMed Scopus Google Scholar and for D. Ovrebo S. Sejersted O.M. Toxicokinetics of formate during hemodialysis.Acta Med Scand. 1983; 214: 409-412Crossref PubMed Scopus (35) Google Scholar and D. McMartin K.E. Methanol and ethylene glycol poisonings.Mechanism of toxicity, clinical course, diagnosis and treatment. Med Toxicol. 1986; 1: 309-334Google Scholar data on the of IHD to D. McMartin K.E. Methanol and ethylene glycol poisonings.Mechanism of toxicity, clinical course, diagnosis and treatment. Med Toxicol. 1986; 1: 309-334Google S. techniques in the treatment of Full Text PDF PubMed Scopus Google Scholar, et of methanol intoxication with hemodialysis an J Dis. 30: Full Text PDF PubMed Scopus Google Scholar, J. et use of an and dialysate to serum ethanol during for methanol PubMed Scopus Google Scholar, et hemodialysis for the treatment of patients with methanol J 2005; Scholar, R. R. et of hemodialysis with for the treatment of alcohol Scholar, G. in the poisoned PubMed Scopus Google Scholar the is scarce for formate D. Ovrebo S. Sejersted O.M. Toxicokinetics of formate during hemodialysis.Acta Med Scand. 1983; 214: 409-412Crossref PubMed Scopus (35) Google P. P. kinetics in methanol Toxicol. 2005; PubMed Scopus Google McMartin et al.Formate kinetics in methanol poisoning.J Toxicol. 2002; 40: PubMed Scopus Google Scholar are studies both the on methanol and formate kinetics with continuous of elimination (CVVHD/HDF) and only that the efficacy of continuous and intermittent of McMartin et al.Formate kinetics in methanol poisoning.J Toxicol. 2002; 40: PubMed Scopus Google Scholar In that were only patients, and formate kinetics was The elimination of methanol therapy is of order with a rate of is of that of D. R. et al.Methanol and formate kinetics in methanol PubMed Scopus Google Scholar methanol is by ethanol methanol elimination is very half-lives of in both by some J. McMartin S. et for the treatment of methanol J Med. PubMed Scopus Google D. Ovrebo S. et of methanol in J 1983; 43: PubMed Google Scholar The kinetics of methanol and the rate kinetics may only on the of toxic but also on the from to of different of ethanol in and of and renal as well as possible and in alcohol and Both half-lives during extracorporeal elimination in study are the elimination half-life of methanol (mean during treatment with antidotes K.E. Andersson K.S. Urdal P. et al.Methanol and formate kinetics during treatment with fomepizole.Clin Toxicol. 2005; 43: 221-227Crossref PubMed Scopus (49) Google et al.Methanol half-life during ethanol for of methanol Med. Full Text Full Text PDF PubMed Scopus Google Scholar results further the of IHD the CVVHD/HDF in methanol elimination from from a G. G. et with intermittent in the treatment of methanol Scopus Google Scholar the circulatory is a when dialysis modality in where is this is why the continuous modalities are for patients with In methanol however, is the cases with the mean arterial blood pressure are patient in and therefore IHD is to be CVVHD/HDF in In IHD patients more because of enhanced due to and of were in 13 cases of treatment with complications were in patients treated with was intermittent The is when the toxic to be the arterial from is the with methanol and formate. The of methanol is in the cases of of methanol from a In the mean to diagnosis was in the IHD and the methanol was before the of The of formate if is to serum ethanol and of the metabolic of methanol elimination in addition to the elimination through In all was by ethanol by was by kinetics of methanol elimination. In to the study by et G. G. et with intermittent in the treatment of methanol Scopus Google Scholar the mean methanol elimination half-life in study was only 54% during IHD as with The the two of dialysis in study be by the higher dialysate flow in the CVVHD/HDF The dialysate flow rate in two patients on in the study was with a mean of in The results for formate elimination during IHD from the elimination half-life of formate from K.E. Andersson K.S. Urdal P. et al.Methanol and formate kinetics during treatment with fomepizole.Clin Toxicol. 2005; 43: 221-227Crossref PubMed Scopus (49) Google Scholar P. P. kinetics in methanol Toxicol. 2005; PubMed Scopus Google McMartin et al.Formate kinetics in methanol poisoning.J Toxicol. 2002; 40: PubMed Scopus Google S. et acid and enhanced renal elimination in formic acid Toxicol. PubMed Scopus Google Scholar to P. P. kinetics in methanol Toxicol. 2005; PubMed Scopus Google McMartin et al.Formate kinetics in methanol poisoning.J Toxicol. 2002; 40: PubMed Scopus Google S. et acid and enhanced renal elimination in formic acid Toxicol. PubMed Scopus Google Scholar and in two S. concentrations in a of methanol 30: Google and K.E. H. Urdal P. et formate elimination in methanol poisoning: a Toxicol. Scopus Google Scholar was also in a study by et McMartin et al.Formate kinetics in methanol poisoning.J Toxicol. 2002; 40: PubMed Scopus Google Scholar during This however, some only patients were and two of data to the half-life of formate (of whom an half-life before of was in the blood flow in two patients of during that half-life be results with of et P. P. kinetics in methanol Toxicol. 2005; PubMed Scopus Google Scholar, the mean formate elimination half-life during hemodialysis in patients to be was statistically different from the values the formate elimination half-life before in the of dialysis in the study was in the studies on The mean elimination half-life of formate on IHD in study was 56% as with CVVHD/HDF vs. 3.6h, and significantly the half-lives in the studies of et McMartin et al.Formate kinetics in methanol poisoning.J Toxicol. 2002; 40: PubMed Scopus Google Scholar and et P. P. kinetics in methanol Toxicol. 2005; PubMed Scopus Google Scholar results that the mean elimination half-life of formate and methanol results from the higher rates of blood flow and dialysate The the blood flow and the elimination half-lives of methanol and formate were 1 and with elimination half-lives with blood flow flow rate was the significant in the for formate and of the significant in the for In the elimination half-lives were when the dialysate flow rate was higher and the was The elimination half-life of formate in two patients treated with the dialysate flow rate possible on was significantly (mean as with the mean for this group the of the blood flow and the dialysate flow when continuous dialysis should be increased as as the on the dialysis this should be performed a evaluation of the and of the We of and formate elimination the by et H. N. S. of visual after methanol Toxicol. PubMed Scopus Google Scholar The in formate elimination half-lives the patients with and acid was statistically In addition, hemodialysis the formate elimination to an that the of the of formate is further studies are to this on the of dialysis in methanol poisoning on serum if of the toxic formic acid are K.E. Urdal P. Jacobsen D. Increased serum formate in the diagnosis of methanol poisoning.J Toxicol. 2005; PubMed Scopus Google Scholar and therefore as the of metabolic and the gap are K.E. Hunderi O.H. Rudberg N. et al.Anion and osmolal gaps in the diagnosis of methanol poisoning: clinical study in 28 patients.Intensive Care Med. 2004; 30: 1842-1846Crossref PubMed Scopus (94) Google Scholar In methanol is the of toxic formate by dialysis and the correction of metabolic that is and the of serum methanol is the if is by ethanol may the clinical that of IHD are in methanol that the metabolic is is and formate is In mass methanol outbreaks with a of patients, if the dialysis are we a of of IHD and of CVVHD/HDF before of the if the methanol concentration the osmolal gap be This all formate and as well as a of the The treatment for after the of the as well as the of of of acidosis, this a and methanol elimination to on data, an for of IHD and of that of serum methanol is is patients should treatment for of IHD and of The with this of the of dialysis is that is the elimination of the toxic study the of IHD CVVHD/HDF in of the rate of elimination of both methanol and formate, the especially in the patients. the of the increased elimination with the higher blood and dialysate we dialysis by the blood flow rate on IHD and the blood and dialysate flow rate on CVVHD/HDF as as the limitations of the and the patient studies with more cases are to whether the of hemodialysis also the and in methanol-poisoned however, data a the rate of formate elimination and of methanol poisoning: the patients with significantly elimination half-lives of formate The limitations of this study be to possible in the and of toxic in the methanol and formate and the available modalities for treatment the modality of enhanced add to the the two groups of patients treated with different of enhanced elimination were by and of the data for all were in laboratory data and both were of data for the IHD patients data however, the was and the on the was This study was as a because the choice of the of enhanced elimination in was on clinical practice as and by different the of this study was as a study the modality in of the in methanol and formate elimination half-lives should be by the above of the The study was a with to the hemodialysis modality of and were if they were of and the methanol poisoning methanol of methanol and to and laboratory data, serum methanol and formate on and the of methanol-poisoned patients were treated in and The patients were treated with ethanol a of both antidotes (for example, of therapy with fomepizole by ethanol were to patients to the were treated with IHD, and patient were treated with continuous methods of enhanced elimination with and with of the patients treated with IHD were admitted to the of and the patients treated with CVVHD/HDF were admitted to the of and hemodialysis if they of the serum methanol higher visual toxicity. The choice of the of enhanced elimination in was on as the of the patient on the patients treated with IHD mean arterial pressure patients treated with mean arterial pressure and of clinical of poisoning patients treated with were on whereas only treated with significant was the of available in a medical the in the only the continuous of hemodialysis The IHD for the ethanol was to patients treated with IHD and patients were treated with whereas fomepizole by ethanol was administered to patients in patients treated with IHD and 11 patients treated with CVVHD/HDF IHD was after to the hospital was performed for a of CVVHD/HDF was started after to the hospital and for a of blood for methanol and formate was obtained on the of during the dialysis and the of The blood for the were the as the for the methanol and formate in this and studies and methanol could be the available was The of CVVHD/HDF were during the In the of CVVHD/HDF was the however, was for the study and to blood were serum was and was Methanol was measured by a gas with and a with Czech of and of and were by of methanol Czech was measured on a formate and to a K.E. Urdal P. Jacobsen D. Increased serum formate in the diagnosis of methanol poisoning.J Toxicol. 2005; PubMed Scopus Google Scholar, with formate of Scholar, P. et enzymatic for plasma Toxicol. Scopus Google Scholar, P. for in as for a 30: Scholar formate was used to a of and two of was and the was Serum ethanol was by gas chromatography with and a with The of was and the of was were Czech was measured by on a The for the osmolal gap was per kg The osmolal from ethanol was from the measured half-life was from the of methanol and formate versus the elimination from the of the of methanol formate versus Clinical Scholar The elimination half-life was then from the The and the osmolal gap were by the - and - where the measured K.S. et and gaps in patients admitted to an emergency medical Toxicol. PubMed Scopus Google Scholar Data are as with The elimination half-lives were the was performed for of concentration of methanol and formate on and were for the of values of different groups of patients. The of data was on sample and the of sample was on the We used the for of two groups of patients, and the and for the of of for the methanol and formate elimination We used also the on the as an of was performed in and the were in Czech and in and (both to of of the in The study was by the in Czech This study was by the of the in the study of of methanol of Czech and the for of of Czech and the and for the of and therapy of and and their significant risk in the in by We and of for the of the