Please find attached the detailed reply to all reviewer s comments/suggestions.

Transcription

1 Author s response to reviews Title: The Impact of the Time of Drug Administration on the Effectiveness of Combined Treatment of Hypercholesterolemia with ROSuvastatin and EZEtimibe (RosEze): study protocol for a randomized controlled trial Authors: Karolina Obońska (kalaobonska@op.pl) Michał Kasprzak (medkas@o2.pl) Joanna Sikora (wojcikiewicz.j@gmail.com) Ewa Obońska (ewaobonska@onet.eu) Krzysztof Racki (krzysztof.racki@gmial.com) Natalia Goździkiewicz (natalia.gozdzikiewicz@gmail.com) Magdalena Krintus (krintus@wp.pl) Jacek Kubica (jkubica@cm.umk.pl) Version: 1 Date: 26 Apr 2017 Author s response to reviews: Dear Dr James A. Betts, We appreciate very much your interest in our manuscript. We have been able to resolve the issues raised by the reviewers, and have made appropriate changes accordingly. Please find attached the detailed reply to all reviewer s comments/suggestions.

2 Changes made in the manuscript are in red or blue or purple. We trust that you will find these changes satisfactory and we look forward to hearing from you at your earliest convenience. Yours sincerely, Karolina Obońska, MD, PhD Reviewer s comments: Reviewer #1: Summary: This manuscript describes the protocol for a registered clinical trial whose aim is to evaluate the impact of the time of day (morning or evening) of administration of a combined statin (Rosuvastatin) and cholesterol absorption inhibitor (Ezetimibe) on total cholesterol, low-density lipoprotein cholesterol (LDL-c) and other secondary outcomes. The study will be conducted over 12 weeks in 200 patients in a randomised crossover manner. All drugs will be open-label and the study will be performed at a single centre. Half the patients will receive the combined therapy in the morning first (0800) and the other half in the evening (2000) for the first 6 weeks before crossover for the final 6 weeks. The study has a registered clinical trial number, has local ethical approval and there are no competing interests by the researchers. Comments: This is an interesting study but there are several points (listed below) related to design, methodology and statistical analysis which are unclear and should be addressed at the outset.

3 Specific Comments: 1. Introduction: At the end of the introduction it states that Rosuvastatin can be administered at any time of day. If this is the case, please justify why you think changing the administration time of the drug under combined therapy is expected to have any different effect. Answer: We modified the introduction accordingly. In our everyday practice we meet many patients with hypercholesterolemia treated with statins. All of them are advised to take statin in the evening,whereas in case of combined treatment with ezetimibe they take the latter in the morning. Page: 6 Line: To fill this evidence gap, the goal of this study is to determine whether time of the day of rosuvastatin and ezetimibe administration plays any role in the effectiveness of the drug, and to identify side effects of combined therapy with rosuvastatin and ezetimibe administered at the same time of the day. Furthermore, we aim to assess whether administration of lipid lowering drugs in the morning improves adherence compared with their evening administration. Page: 7 Line: Introduction: It would be worthwhile hypothesising what you are expecting to observe. Answer: We modified the introduction accordingly. Considering the potency of rosuvastatin further enhanced when administered in combination with ezetimibe, we expect significant reduction of LDL cholesterol concentration. However the role of the time of drug administration in this case is questionable and worth further evaluation. Page: 7 Line: 9-12

4 3. Participants: It is not stated who this study is aimed at testing? At present it states 200 adult patients with hypercholesterolemia, as defined by European guidelines, who have had ineffective statin monotherapy for their cholesterol treatment for at least 6 weeks. Is this study looking at cholesterol in individuals in a primary prevention setting or secondary prevention setting? The outcomes may be very different. The introduction talks about both settings and this is rather confusing. Answer: Thank you for that comment. At the time the study was in a design phase, we intended to include both patients in a primary as well as in secondary prevention setting. However, while the recruitment has eventually started we decided to include only individuals in secondary prevention setting. All study participants were on statin therapy due to secondary prevention indications. Page: 7 Line: Please define 'ineffective statin monotherapy' after 6 or more weeks. Is this defined as not meeting the total cholesterol/ldl-c goal or an actual or stated percentage change in these outcomes over 6 weeks? Answer: Thank you for that suggestion. Patients enrolled into the study were found eligible for the study when despite statin monotherapy the LDL-C concentration was higher than 70 md/dl. Page: 7 Line: Methods: Statin monotherapy, one assumes, means any statin treatment which has been ineffective? Thus, you will have two different treatment groups. The first have already been taking Rosuvastatin and will then move into the combined treatment. The second would be taking other forms of statin and then move into the combined treatment. Thus, any change in the first group one may argue is simply from the administration of Ezetimibe, whereas changes in the second result from the combined administration.

5 Answer: Thank you for that comment. That is correct, that we enroll all patients with ineffective statin treatment, regardless which statin was used. The use of rosuvastatin was not exclusion criterion. If the patient was given rosuvastatin before the enrollment, the LDL cholesterol will be reduced only due to ezetimibe co-administration. However, it will be only one subgroup of patients. Because after the recruitment is completed, we will analyze all patients, as well as possible subgroups of patients. 6. Methods: As noted in the previous point, if Rosuvastatin therapy has been ineffective then any change resulting in the course of this study may be attributed to the effect of Ezetimibe alone unless an Ezetimibe only arm were added. Answer: Thank you for that comment. We did not want to exclude patients using rosuvastatin, because it will be very interesting to assess effectiveness and tolerance of combined treatment with intermediate dose of rosuvastatin (10 mg) and ezetimibe in patients ineffectively treated with rosuvastatin administered alone. There was not such a study provided in Polish population. We do not consider toadd Ezetimibe only arm. 7. Methods: Blood sampling will be taken in the morning after a 12 hour fast. How will this be affected by use of medication? Presumably the last use of medication could vary from 12 hours for the evening group or 0 to 24 hours for the morning group depending if they take the last medication on the morning of the sample or the morning prior to the sample. Answer: Thank you for that remark. However in our study each patient will be tested twice: once after 12 hours from the last dose of rosuvastatin/ezetimibe and once after 24 hours from the last drug dose. One of the study objects is whether time of the day of the drug administration has influence on its effectiveness. 8. Methods: There is no washout period. It is worth noting that this will likely obscure differences between the two groups unless one time of day of administration is completely ineffective or causes a substantial regression or improvement in one group.

6 Answer: Thank you for that comment. This is a potential limitation of our study and certainly will be mentioned in an appropriate section of further publication. After the study will be completed we will analyze whether the is or no any influence of the time of the drug administration on effectiveness of the drug. 9. Statistical analysis: There is no power analysis provided - why? What is the expected change in the groups? What is considered the smallest worthwhile change at the outset? Answer: Thank you for that remark. We corrected the statistical analysis paragraph accordingly. Since there is no reference study examining the effectiveness of combined treatment of hypercholesterolemia with rosuvastatin and ezetimibe according to timing of drug administration, we decided to perform an internal pilot study of 20 patients to estimate the final sample size. The means and standard deviations of reduction in LDL-C was ± mg/dl and ± mg/dl during morning and evening administration respectively. The correlation coefficient between total cholesterol reduction during morning and evening drug administration was Based on this results and assuming a two-sided alpha value of we calculated using the t-test for dependent variables, that enrollment of 157 patients would provide an 98% power to demonstrate a significant difference in total cholesterol level. To compensate for potential withdrawal of consent or loss of study participants due to other reasons, we plan to enroll 200 patients. Page 11 Line 22-24and Page 12 Line Methods/Statistical analysis: There is no mention of how external factors will be monitored and encompassed into the analysis. These include changes in physical activity, diet and body mass. Answer: Thank you for the comment. Patients enrolled into our study are already treated for hypercholesterolemia for a certain period of time. We can assume that lifestyle changes were not effective. However, we plan to monitor their weight during the study, as well as provide a survey according their diet and physical activity on every visit.

7 11. Statistical analysis: It is noted that the therapy is provided free of charge. Nevertheless, please detail how you will document and treat non-compliance and also study dropout. If these individuals are patients there may also be issues with cardiovascular events or morbidity. Answer: All patients during enrollment visit and then on the second visit are given 42 tablets of study drug. On the second and third visit they are obligated to return empty boxes and any tablets left. If the patient will not be present at the second or third visit we contact with them or their families by telephone. 12. Figure 1: It would be useful to indicate the number of individuals in each group. Also the time of administration of the treatment on the Figure rather than just morning or evening. Answer: We modified the figure accordingly. Reviewer #2: General Comments Although the general structure of this study comes through a few extra details would be useful for the reader. In the introduction it is clear that statins have been shown to be effective and that this combined therapy may improve adherence, but there is no indication of the scale of difference that might be seen. As similar stats have been given for the efficacy of statins and combined therapy it would be good to extend this to cover the potential of time of day to create a stronger rationale. Similarly, in the methods other details could be included to help the reader comprehend the study, such as exactly how your comparison of the two timings will be structured, how you established your target sample size and how measurements of your key endpoints will actually be obtained.

8 Specific Comments Abstract: Page 3, Line 35/36 - This sentence does not read correctly, consider "In order to prevent nonadherence to the treatment, all patients will receive the study drug for free". I also think that stating that this will fully prevent non-adherence is excessive, it will certainly help to minimise but not fully prevent as this is also influenced by factors other than cost. Answer: Thank you for that comment. We modified the abstract as well as methods accordingly. Page 3, Line Page 3, Line 40/41 - It seems somewhat contradictory to have total cholesterol listed as a primary outcome. Although total cholesterol will certainly be influenced by changes in LDL-C concentration, it is also influenced by HDL-C concentration which is listed as a secondary outcome. I think it would provide clarity if you have LDL-C as your single primary outcome, which would be in keeping with the theme in the introduction, and add total cholesterol to the your list of secondary outcomes. Answer: Thank you for that remark. We modified the abstract and methodssection accordingly. Page 3, Line 17 and 19 Background: Page 5, Line 15/16 - A reference is needed to back up this point, in addition a statistic on the failure rate of lifestyle interventions in resolving hypercholesterolemia would strengthen your rationale. Answer: We corrected the background section accordingly. Page 5, Line 5,6

9 Page 5, Line 22/23 - The sentence "When the low-density lipoprotein cholesterol (LDL-C) goal is not achieved, combination of statin with a cholesterol absorption inhibitor - ezetimibe may be considered (class of recommendation IIa, level of evidence B) [2]." should be moved to line 49/50 before the sentence beginning "Statin dose titration seems to ". This should improve flow by presenting the problem, followed by the effectiveness of current therapies, and lastly what is done when this does not succeed. Answer: We corrected the background section accordingly. Page 5, Line Page 5, Line 46/47 - Again, a reference is needed to support the statement "Despite intensive statin therapy provided, a large group of patients still does not reach therapeutic goals". It would also be useful to include a statistic on the success/failure rate. Answer: Thank you for that comment. We corrected this paragraph accordingly. Page 5, Line Page 6, Line 37/38 - The paper creates a strong argument for studying ROSuvastatin and highlights how the results could impact adherence, but there is a distinct lack of literature on how time of day has been shown to influence effectiveness. As such it does not outline whether we are expecting a 2% improvement or a 20% improvement which is a key part of the rationale for this. Although, as stated, there are "no studies provided assessing the effectiveness of combined treatment of hypercholesterolemia with rosuvastatin and ezetimibe according to timing of drug administration", it was mentioned that the impact of statins is modulated by time of administration so even some values on this would provide an indication. Answer: Thank you for that remark. However, while preparing this publication we searched literature regarding evidence supporting morning vs evening administration of rosuvastatin or rosuvastatin combined with ezetimibe. Unfortunately the data are very limited. Studies assessing this issue, with the use of other statins, show rather lack of difference between morning vs evening administration of the combined treatment. That is why this problem needs further investigation. Considering the potency of rosuvastatin, further enhanced when especially administered in combination with ezetimibe, we expect significant reduction of LDL cholesterol concentration. However the role of the time of the day of the drug administration in this case is questionable and worth further evaluation.

10 Page 7, Line 9-12 Methods: Page 7, Line 2/3 - Some of the exclusion criteria are quite specific so some indication of how this is screened for would be useful (i.e. is it self-reported, taken from medical records, etc). In addition, how will these participants be recruited? Answer: The recruitment will be carried out among patients admitted to the Cardiology Department of University Hospital no 1 in Bydgoszcz. Information according the exclusion criteria will be obtained from eligible patients based on their report as well as from medical records obtained during hospitalization. Page 7, Line 25/26 - How will the randomization be done? Answer: Randomization is carried out as follows: after the eligible patient is recruited into the study physician draws one of the enclosed envelopes containing the name of the arm to which the patient is enrolled to. Page 7, Line 35/36 - The wording could be improved here, try: "In order to encourage adherence to the treatment, all patients will receive the study drug for the entire observational period for free." - This will also address the point raised in the abstract about "preventing non-adherence". Answer: Thank you for your comment. We corrected the sentence accordingly. Page 8, Line Page 7, Line 40/41 - Please provide details of how a sample size of 200 was decided upon (i.e. sample size/power calculation), as it is it seems to be an arbitrary number. Answer: Thank you for that remark. We corrected the statistical analysis paragraph accordingly. Since there is no reference study examining the effectiveness of combined treatment of hypercholesterolemia with rosuvastatin and ezetimibe according to timing of drug administration, we decided to perform an internal pilot study of 20 patients to estimate the final

11 sample size. The means and standard deviations of reduction in LDL-C was ± mg/dl and ± mg/dl during morning and evening administration respectively. The correlation coefficient between total cholesterol reduction during morning and evening drug administration was Based on this results and assuming a two-sided alpha value of we calculated using the t-test for dependent variables, that enrollment of 157 patients would provide an 98% power to demonstrate a significant difference in total cholesterol level. To compensate for potential withdrawal of consent or loss of study participants due to other reasons, we plan to enroll 200 patients. Page 11 Line and Page 12 Line 1-8 Page 7, Line 49/50 - It is not clear exactly how the key comparisons will be structured, is it looking at the difference between 6 and 12 weeks, or is it looking at the change over each 6 week block (i.e. week 0 vs week 6, week 6 vs week 12)? If it is the latter then why not include a washout period as any positive effects gained in the first 6 weeks of treatment will be carried over to the opposing arm? I think I know how you must be structuring the comparison but some clarification of this in the text would be useful for the reader. Answer: Thank you for that comment. We will compare the change of LDL cholesterol between first visit (week 0) and after 6 weeks as well as between visit number 1 and 3 (after 12weeks). Moreover, the comparison between visit 2 and 3 will be performed. The washout period would be desirable, but we plan to enroll patients under secondary prevention, that is why we do not plan to stop statin administration. Page 8, Line Page 7, Line 49/50 - As discussed for the abstract, consider making total cholesterol a secondary endpoint given that it is also affected by change in HDL-C which is listed as a secondary endpoint. Answer: We corrected theendpoints paragraph accordingly. Page 9, Line 1 Page 8, Line 10/11 - Some clarification on why some of these secondary endpoints are being measured would be additive.

12 Answer: Thank you for that remark. Our secondary endpoints include all lipid fractions, as well as liver enzymes and markers of muscle damage. Furthermore, we assess glucose metabolism parameters, because patients are provided with repeated dietary advice during scheduled visits, that may have influence on better diabetes control. High sensitivity C - reactive proteinare evaluated to assess, whether potent statin as rosuvastatin decrease concentration of inflammatory markers. At last, plasma fluorescence assessment using stationary and time-resolved fluorescence spectroscopy at pre-defined time points are evaluated because our previous unpublished studies confirmed that plasma from patients with high triglyceride concentration has specific fluorescence lifetime. We decided to continue our observations using plasma from patients treated with rosuvastatin and ezetimibe. Page 8, Line 44/45 - Pre-trial standardisation seems very minimal here, are there no other restrictions on exercise, alcohol, caffeine, etc? Answer: Thank you for that comment. Patients are also advisedto abstain from alcohol and avoid excessive physical effort within 48 hours preceding the blood collection. Page 10, Line 5-7 Page 8, Line 49/50 - How will these parameters be evaluated, ELISA? model/manufacturer details for any automated analysers? Answer: Thank you for that comment. The suggested information were added to the text. Following centrifugation routine laboratory measurements are performed in fresh serum (glucose, creatinine, basic lipid profile [total cholesterol, LDL-C, HDL-C, TG], AST, ALT, GGT, CK) and only HbA1c is measured in whole blood. All remaining serum is aliquoted and stored at -80 C until assayed for hs-crp, apoai, apob, Lp(a) and sd-ldl-c. All measurements (except for HbA1c) are performed on the Horiba ABX Pentra 400 analyzer (Horiba ABX, Montpellier, France). LDL-C is measured directly and non-hdl-c is calculated. Reagents for Lp(a) and sd-ldl-c (direct automated sdldl-c kit) are supplied by Randox Laboratories (Crumlin, UK). HbA1c is measured on the BIO-RAD D-10 Hemoglobin Testing System using High-Performance Liquid Chromatography (HPLC). Page 10, Line 9-18

13 Page 9, Line 2/3 - Please provide centrifuge temperature and report speed in x g rather than rpm. Also, usually serum samples are allowed to clot before centrifuging, is this not the case in your study? Answer: Thank you for that comment. The suggested information were added to the text. Serum tubes are allowed to clot for 30 minutes in a vertical position at room temperature. Serum is separated from venous blood samples by centrifugation for 10 minutes at 3000xg at room temperature. Page 10, Line 7-9 Page 9, Line 14/15 - Which measures are actually being taken from serum and as with the previous paragraph how are they being obtained (e.g. by ELISA or another method)? Answer: Thank you for your comment. We added suggested information to the text. All measurements (except for HbA1c) are performed on the Horiba ABX Pentra 400 analyzer (Horiba ABX, Montpellier, France). LDL-C is measured directly and non-hdl-c is calculated. Reagents for Lp(a) and sd-ldl-c (direct automated sdldl-c kit) are supplied by Randox Laboratories (Crumlin, UK). HbA1c is measured on the BIO-RAD D-10 Hemoglobin Testing System using High-Performance Liquid Chromatography (HPLC). Page 10, Line Page 9, Line 20/21 - As before, report centrifuge speed in x g and give the temperature this was done at please. Answer: Thank you for that comment. The suggested information were added to the text. Plasma is separated from venous blood samples by centrifugation for 10 minutes at 3000xg at room temperature. Page 11, Line 7-8 Page 9, Line 30/31 - This is better, it is clear how these parameters are being measured and model/manufacturer details are provided. Answer: Thank you for that comment. We described in details all measurements.

14 Page 10, Line 7-18and Page 11 Line Page 10, Line 1 - Please clearly state how the comparisons will be structured here (i.e. is it week 6 vs week 12, or week 0 vs week 6 and week 0 vs week 12, or week 0 vs week 6 vs week 12?). Answer: We will perform following comparisons: 1. results from baseline measurement vs results from the measurement after 6 weeks of therapy (0 vs 6 week); 2. results from baseline measurement vs results from the measurement after 12 weeks of therapy (0 vs 12 week); 3. results from the measurement after 6 weeks of therapy vs results from the measurement after 12 weeks of therapy (6 vs 12 week). Page 10, Line 7/8 - Which qualitative variables will there be, none have been clearly outline in the methods provided so far. Does this relate to adherence? Be more specific to make sure the reader knows all the variables that are being measured. Answer: Apart from the analysis in the whole population, all end-points will be analyzed in subgroups depending on age, sex, and presence of other co-morbidities. Page 9, Line Page 10, Line 44/45 - This final sentence does not read properly. Try "Considering that most medications are taken in the morning, it is possible that compliance with administration targets will improve if an effective dose can be taken in the morning instead of the evening." Answer: Thank you for that suggestion. We changed that sentence accordingly.

15 Page 13, Line 6-8 Page 11, Line 42/43 - Typographical error, change "concent" to consent. Answer: Thank you for that suggestion. We changed that word accordingly. Page 14, Line 7 Figure 1: Add the specific time of day in brackets after the statement (e.g. "Drug administered in the morning (08:00)"). Answer: Thank you for that suggestion. We changed the figure accordingly.