A Validated LC–MS/MS Assay for the Simultaneous Quantification of the FDA-Approved Anticancer Mixture (Encorafenib and Binimetinib): Metabolic Stability Estimation
Round 1
Reviewer 1 Report
Authors have addressed most of the comments in the revised version of the manuscript. However, there are two points that are still unclear:
- please see my previous comment #2: It is not clear yet that under which setting this method would be utilized? i.e. will this method be utilized in clinics, patients, etc and to monitor, diagnose etc? What would be the utility of this method?
- please see my previous comment #3: Authors have now included both citations in the revised manuscript. Authors added to their manuscript that
"There is only one published article on the simultaneous analysis of BNB and ENF in pure pharmaceutical ingredients
and formulations, but the linearity was 2–20 and 6–20 μg/mL for BNB and ENF, respectively, which is not enough for the required limit for metabolic stability studies"
since the method is already published and difference was the linearity as stated by the authors. This states that the method developed in the manuscript is an improved version of the method that has already been reported before, which reduces the novelty of the method presented in this manuscript.
Author Response
Reviewer 1
Authors have addressed most of the comments in the revised version of the manuscript. However, there are two points that are still unclear:
please see my previous comment #2: It is not clear yet that under which setting this method would be utilized? i.e. will this method be utilized in clinics, patients, etc and to monitor, diagnose etc? What would be the utility of this method?
Reply:
- This method could be applied for studying the metabolic stability of ENF and the metabolic stability of BNB either alone or in combination. Also it could be utilized for studying the drug-drug interaction between the two drugs on the metabolic stability.
- We approved that the developed analytical method (LC-MS/MS) could be utilized for determination of BNB and ENF in human plasma and urine as seen in the attached supplementary material. We uploaded this as supplementary and we gave a hint in the manuscript about that but this is not the mean target of the manuscript as it is designed for metabolic stability study for BNB and ENF that should involve only human liver microsomes matrix.
please see my previous comment #3: Authors have now included both citations in the revised manuscript. Authors added to their manuscript that
"There is only one published article on the simultaneous analysis of BNB and ENF in pure pharmaceutical ingredients and formulations, but the linearity was 2–20 and 6–20 μg/mL for BNB and ENF, respectively, which is not enough for the required limit for metabolic stability studies"since the method is already published and difference was the linearity as stated by the authors. This states that the method developed in the manuscript is an improved version of the method that has already been reported before, which reduces the novelty of the method presented in this manuscript.
Reply:
- This study describes a new analytical LC-MS/MS method for the concurrent quantification of encorafenib (Braftovi™ [ENF]) and binimetinib (Mektovi® [BNB]), which are important drugs for the treatment of unresectable melanoma. We believe that our study makes a significant contribution to the literature because in addition to describing a new method of measurement for encorafenib and binimetinib, this study also evaluated the metabolic stabilities of encorafenib and binimetinib in a human liver microsome matrix. We found that both drugs undergo moderate clearance from the blood by the liver, which indicates good in vivo In silico metabolic vulnerability assessment of ENF and BNB were performed utilizing P450 module of StarDrop software.
- We extend the application of the developed LC-MS/MS method for quantification of these analytes in biological fluids using human urine and plasma explaining the protein precipitation as a method of extraction.
Author Response File: Author response Reviewer 1
Reviewer 2 Report
As far as I remember, this work was already presented under reference molecules-1024636. My comments were “The manuscript is rather well constructed and well documented on the validation point of view. However, the overall interest of this paper and its contribution to the literature look weak for several reasons” and my opinion did not change although the authors brought some improvement. As already mentioned in the first evaluation of this work, “Molecules”, which Impact Factor is higher that 3.5 (5-Year Impact Factor), deserves better than the publication of a ordinary validation report.
Author Response
Reviewer 2
- As far as I remember, this work was already presented under reference molecules-1024636.
Reply
Yes, this work was already submitted (molecules-1024636), and this is a resubmission after addressing the reviewer’s comments as was directed by the editor.
- My comments were “The manuscript is rather well constructed and well documented on the validation point of view. However, the overall interest of this paper and its contribution to the literature look weak for several reasons” and my opinion did not change although the authors brought some improvement. As already mentioned in the first evaluation of this work, “Molecules”, which Impact Factor is higher that 3.5 (5-Year Impact Factor), deserves better than the publication of ordinary validation report.
Reply
We approved the outcomes and the application of this work by quantification of the two drugs in biological fluids (ex. Human plasma) using protein precipitation method by adding double volume of ACN and data was attached as a supplementary.
This study describes a new analytical method for the concurrent quantification of encorafenib (Braftovi™ [ENF]) and binimetinib (Mektovi® [BNB]), which are important drugs for the treatment of unresectable melanoma. We believe that our study makes a significant contribution to the literature because in addition to describing a new method of measurement for encorafenib and binimetinib, this study also evaluated the metabolic stabilities of encorafenib and binimetinib in a human liver microsome matrix. We found that both drugs undergo moderate clearance from the blood by the liver, which indicates good in vivo bioavailability. In silico metabolic vulnerability assessment of ENF and BNB were performed utilizing P450 module of StarDrop software.
Author Response File: Author Response Reviewer 2
Reviewer 3 Report
In this work, the authors reported a sensitive and rapid LC-MS/MS method for the simultaneous estimation of encorafenib and binimetinib. This is an interesting work because such a method is probably useful for calculating intrinsic clearance and in vitro half-life for metabolic stability assessments. Following are some suggestions or comments to this manuscript:
1. All the abbreviations, such as RAS, RAF, MEK, and ERK should be defined at their first mention in the manuscript.
2. Figure 4: It is not easy to understand this result. Please note the curves with different colors.
3. Overall, this manuscript looks like a test report rather than a research paper since the authors only presented their own results without any discussion and comparison with the previous publications. Maybe the authors would justify that there is only one published article on the simultaneous analysis of BNB and ENF in pure pharmaceutical ingredients and formulations. However, to the best of my knowledge, many similar studies and publications in this field can be easily found and must be included and discussed in this paper. For example,
Huynh, Huu H., et al. "Development and validation of a simultaneous quantification method of 14 tyrosine kinase inhibitors in human plasma using LC-MS/MS." Therapeutic drug monitoring, 39 (2017): 43-54.
Jiang, Hao, et al. "Fully validated LC-MS/MS assay for the simultaneous quantitation of coadministered therapeutic antibodies in cynomolgus monkey serum." Analytical Chemistry, 85 (2013): 9859-9867.
Van Erp, Nielka P., et al. "A validated assay for the simultaneous quantification of six tyrosine kinase inhibitors and two active metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry." Journal of Chromatography B, 937 (2013): 33-43.
Please add a Discussion section.
Author Response
Reviewer 3
In this work, the authors reported a sensitive and rapid LC-MS/MS method for the simultaneous estimation of encorafenib and binimetinib. This is an interesting work because such a method is probably useful for calculating intrinsic clearance and in vitro half-life for metabolic stability assessments. Following are some suggestions or comments to this manuscript:
- All the abbreviations, such as RAS, RAF, MEK, and ERK should be defined at their first mention in the manuscript.
Reply:
All abbreviations were defined at their first appearance:
Ex, This pathway's activation generates a signal cascade that results in sequential phosphorylation and activation of MAPK kinases such as Rat sarcoma (RAS), serine/threonine kinases Rapidly Accelerating Fibrosarcoma (RAF), extracellular signal-regulated kinase (ERK) and MAPK/ERK kinase (MEK).
- Figure 4: It is not easy to understand this result. Please note the curves with different colors.
Reply:
This is an automatic generated figure from the processing software. We clarified the different color curves in the figure caption.
- Overall, this manuscript looks like a test report rather than a research paper since the authors only presented their own results without any discussion and comparison with the previous publications. Maybe the authors would justify that there is only one published article on the simultaneous analysis of BNB and ENF in pure pharmaceutical ingredients and formulations. However, to the best of my knowledge, many similar studies and publications in this field can be easily found and must be included and discussed in this paper. For example,
- Huynh, H. H.; Pressiat, C.; Sauvageon, H.; Madelaine, I.; Maslanka, P.; Lebbé, C.; Thieblemont, C.; Goldwirt, L.; Mourah, S., Development and Validation of a Simultaneous Quantification Method of 14 Tyrosine Kinase Inhibitors in Human Plasma Using LC-MS/MS. Ther Drug Monit 2017, 39, (1), 43-54.
- Jiang, H.; Zeng, J.; Titsch, C.; Voronin, K.; Akinsanya, B.; Luo, L.; Shen, H.; Desai, D. D.; Allentoff, A.; Aubry, A. F.; Desilva, B. S.; Arnold, M. E., Fully validated LC-MS/MS assay for the simultaneous quantitation of coadministered therapeutic antibodies in cynomolgus monkey serum. Anal Chem 2013, 85, (20), 9859-67.
- van Erp, N. P.; de Wit, D.; Guchelaar, H. J.; Gelderblom, H.; Hessing, T. J.; Hartigh, J., A validated assay for the simultaneous quantification of six tyrosine kinase inhibitors and two active metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2013, 937, 33-43.
Reply
We cited these publications mentioning the importance of using LC-MS/MS technique in quantifying these analytes in different matrices.
Thus, an appropriate and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous detection and quantification of the BNB and ENF in the biological matrix (human liver microsomes) was developed to achieve the required quantification limit [20-22].
4- Please add a Discussion section.
Reply
The following part was updated in the revised version of the manuscript:
A validated LC-MS/MS method was developed for the simultaneous estimation of ENF and BNB that is characterized by sensitivity, rapidity (run time = 6 min), high recovery, and accuracy. This method was applied for studying the metabolic stability of ENF and BNB. The ENF and BNB metabolic stability calculations showed moderate CLint (16.09 and 11.49 µL/min/mg) and in vitro t1/2 (43.1 and 60.3 min). Our calculations showed that when ENF and BNB are used concurrently, ENF is metabolized slightly slower, and BNB is metabolized slightly faster (Table 5). ENF is primarily metabolized through the CYP3A4 catalyzed phase I metabolism. In contrast, BNB is primarily metabolized by the UGT1A1 catalyzed conjugation and, to a lesser extent, by CYP1A2 and 2C19 catalyzed phase I metabolism [6–8], supported our results of no significant effect on the metabolic stability of ENF and BNB when co-administered. These data and other parameters could also predict BNB and ENF in vivo pharmacokinetics using the Cloe PK simulation software.
Furthermore, there doesn’t appear to be any significant influence of ENF or BNB on each other's metabolic stability or metabolic disposition when used concurrently; consequently, it is unnecessary to recalculate doses for concurrent use of ENF and BNB. From metabolic stability data, we can advise that plasma levels should be measured in cases where these drugs are used together since they can accumulate to toxic levels.
Author Response File: Author Response Reviewer 3
Reviewer 4 Report
The current manuscript describes the metabolic stability of two anticancer drugs encorafenib and binimetinib by LC-MS/MS. The subject is interesting and the work done is substantial, well-designed and executed, well documented and reported, whereas the statistical treatment is adequate. A couple of minor issues should be taken into account.
page 1 achieved chromatographic separation of ENF, BNB, and avitinib (an
internal standard) using an isocratic mobile phase on a Hypersil BDS C18 column. probably a typo, please check this out
page 2. of a molecule called BRAF (mutated form). BNB stops a MEK
molecule; please use the mutated form of BRAF protein and the same for MEK
figure 3 please extend the chromatographic span in order to include the molecular ion
please specify the standard error of the slope and the intercept for all analytes and all calibration equation as well as please use one digit after the last 9
2.3.2 the authors use six replicates but the calibration curve includes 12 points please explain
3.5 Generally the use of QC's at the same levels of the calibration curve is not recommended Please comment
Author Response
Reviewer 4
The current manuscript describes the metabolic stability of two anticancer drugs encorafenib and binimetinib by LC-MS/MS. The subject is interesting and the work done is substantial, well-designed and executed, well documented and reported, whereas the statistical treatment is adequate. A couple of minor issues should be taken into account.
Page 1 achieved chromatographic separation of ENF, BNB, and avitinib (an internal standard) using an isocratic mobile phase on a Hypersil BDS C18 column. probably a typo, please check this out
Reply:
The sentence was corrected as (Chromatographic separation of ENF, BNB, and avitinib (an internal standard) was achieved using an isocratic mobile phase on a Hypersil BDS C18 column).
Page 2 of a molecule called BRAF (mutated form). BNB stops a MEK
molecule; please use the mutated form of BRAF protein and the same for MEK.
Reply:
Done:
ENF and BNB blocks the activity of the mutated forms of BRAF and MEK proteins molecules, respectively that are very important in cell growth regulation.
Figure 3 please extend the chromatographic span in order to include the molecular ion
Reply:
These figure is MRM mass transitions that showed only the chosen fragment ions
Please specify the standard error of the slope and the intercept for all analytes and all calibration equation as well as please use one digit after the last 9
Reply:
This was updated in the revised version:
The regression equation for the ENF calibration curve was y = 2.3299x - 4.5808 (R² = 0.9998). The standard error of the slope and intercept were 0.007089 and 1.501, respectively. The regression equation for the BNB calibration curve was y = 0.5287x - 2.2924 (R² = 0.9997). The standard error of the slope and intercept were 0.0006829 and 0.1446, respectively.
2.3.2 the authors use six replicates but the calibration curve includes 12 points please explain
Reply:
We prepared six analytes standards including calibration standards (ten levels) and quality controls (three levels) as seen in table 1. We corrected this error and we clarified this in the revised version:
We performed back-calculations of the six calibration curves (calibration standards) and quality control (QC) samples of ENF and BNB in the HLM matrix, which showed good performance of the methodology. The results of the six calibration curves were used for linearity confirmation and intra-day validation.
3.5 Generally the use of QC's at the same levels of the calibration curve is not recommended Please comment
Reply:
Thirteen standards were used, ten of them were used as calibration standards and three were selected as quality controls. We clarified this in the revised version. We marked quality controls in table 1. So calibration curve standards are different from quality control levels.
Author Response File: Author Response Reviewer 4
Round 2
Reviewer 2 Report
Dear authors,
As already mentioned in my previous reports, I do not have anything concerning your work on a validation point of view. Despite the arguments you put forward, I maintained that the overall interest of this paper and its contribution to the literature is weak as it stand.
You answered that you developed “a new method of measurement for encorafenib and binimetinib”. That is the point. In my first evaluation of your manuscript (referenced as Molecules-1024636), I wrote “the described method adds nothing new to the literature as many validated LC-MS/MS methods have been already described in the recent past concerning this kind of drugs. The assays of benimetinib in human plasma have been validated by UPLC-MS/MS among 2 BRAF inhibitors and two other MECK inhibitors in 2017 [M. Rousset et al]. The assay of Encorafenib by LC-MS/MS in human plasma have been validated among EGFR inhibitors in 2016 [R.W. Sparidans et al]. It cannot be said that the development and the validation of the assay of only these two active molecules by LC-MS/MS is something original compared to the previous cited references. In addition, the authors presents LOQs of 5 ng/mL which are comparable to already published results”.
To summarize my thinking, the development of analytical method for the quantification of encorafenib and binimetinib using reversed phase liquid chromatography is not new, their quantification by tandem mass spectrometry was already published, and similar LOQ have been already reached in the past. Putting the encorafenib and binimetinib all together in a same RPLC chromatogram is not challenging and will not contribute to a so-called “Scientific Soundness” of the paper. However, the biggest part of the discussion in your manuscript is focused on this trivial method whereas the interesting results are somewhere else.
In your answer, you added that “this study also evaluated the metabolic stabilities of encorafenib and binimetinib in a human liver microsome matrix”. You found that “both drugs undergo moderate clearance from the blood by the liver, which indicates good in vivo bioavailability. In silico metabolic vulnerability assessment of ENF and BNB were performed utilizing P450 module of StarDrop software”. In my previous report, I wrote (still in Molecules-1024636) that the results of in silico ENF and BNB metabolic vulnerability were very interesting but not really connected to the discussion. I concluded that this could be an original way to improve this manuscript. However, there are still only 5 lines on the “in silico ENF and BNB metabolic stability” (and Figure 2 which is poorly commented) in paragraph 2.1 and paragraph 2.4 is not connected enough to paragraph 2.1.
To gain in originality, scientific soundness and probably in interest for the reader, I suggest the authors to invert the proportion for each part in their manuscript: summarize the results on validation method and develop the discussion about the metabolic stability.
Author Response
Dear reviewer,
Thank you very much for your efforts in revising our manuscript giving us the chance to improve the way we are thinking.
We updated the manuscript in all parts with the required information including abstract, introduction, results and conclusion focusing on the importance of metabolic stability in drug design. All changes were highlighted in yellow color.
Best regards.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Specific comments concerning the manuscript entitled “A validated LC-MS/MS assay for the simultaneous quantification of the FDA approved anticancer mixture (encorafenib and binimetinib): Metabolic stability estimation” under reference molecules-1024636.
The manuscript is rather well constructed and well documented on the validation point of view. However, the overall interest of this paper and its contribution to the literature look weak for several reasons:
- The authors mentioned in their highlight “Simultaneous assay of Encorafenib and Binimetinib by a validated LC-MS/MS method”. However, the described method adds nothing new to the literature as many validated LC-MS/MS methods have been already described in the recent past concerning this kind of drugs. The assays of benimetinib in human plasma have been validated by M. Rousset et al. by UPLC-MS/MS among 2 BRAF inhibitors and two other MECK inhibitors in 2017 [M. Rousset et al. Clin. Chim. Acta 470 (2017) 8–13]. The assay of Encorafenib by LC-MS/MS in human plasma have been validated by R.W. Sparidans et al. among EGFR inhibitors in 2016 [R.W. Sparidans et al. J. Chromatogr. B 1033-1034 (2016) 390–398]. It cannot be said that the development and the validation of the assay of only these two active molecules by LC-MS/MS is something original compared to the previous cited references. In addition, the authors presents LOQs of 5 ng/mL which are comparable to already published results. Curiously, the authors did not compared their results to the evoked papers.
- The paragraph entitled “Results of in silico ENF and BNB metabolic vulnerability” is very interesting but is not really connected to the discussion. What would be interesting is to check if these molecular structures are effectively observed during the study of the stability of encorafenib and binimetinib. This could be an original way to improve this manuscript.
- There are many problem with the figure 5. The initial protonation site of the molecular ion of BNB, AVB and ENF is not consistent with the structure of their fragments unless the authors can explain the proton shift it implies. Moreover, they indicated m/z 433 for one fragment of AVB and its mass spectrum shows m/z 434.
Other minor points:
- Tables 2, 3, 4 and 5: The significant digits need to be checked. For example, it cannot be written 104.31 % for the accuracy (line 2 table 3) when its RSD is 2.43%. Please, write instead 104 %.
- Table 1 appears in the last position in the manuscript.
- The coefficient “r²” is “determination” not “correlation” as written lines 256, 348.
To conclude “Molecules”, which Impact Factor is higher that 3.5 (5-Year Impact Factor), deserves better than the publication of a simple validation report. This work needs to be improved before it can be accepted as a research publication.
Reviewer 2 Report
This is an interesting study into the metabolism of encorafenib and binimetinib with the help of analytical method development. The study is well designed and the results are well presented. My only comment is about the paper formatting that does not fit the journal requirements.
Reviewer 3 Report
The method presented in this manuscript reads fairly well. The LC-MS/MS data is also presented satisfactorily to some extent. However, there are some major concerns in this manuscript that need to be addressed before publication.
1) Lines 45-48: These lines are unclear. Author first states that there was some increase/decrease in the metabolic clearance when given concurrently, and then in the next statement they state that these drugs do not affect metabolic disposition of each other. These statements are opposite in meaning. Please explain?
2) It is unclear what would be the utility of the method developed here.
3) There are two similar studies that were published recently. How is the method/results presented in this manuscript are different than the already published studies. It is recommended that authors cite these published studies and provide a valid reasoning that how this manuscript compares to the data already published.
Published articles:
1) Stability-indicating UPLC Method for Simultaneous Analysis of Protein Kinase Inhibitors, Binimetinib, and Encorafenib in Pure Active Pharmaceutical Ingredient (API) and Formulation
Lat. Am. J. Pharm. 39 (7): 1428-36 (2020)
http://www.latamjpharm.org/resumenes/39/7/LAJOP_39_7_1_21.pdf
2) Stability indicating UPLC method for simultaneous determination of atorvastatin, fenofibrate and their degradation products in tablets
Journal of Pharmaceutical and Biomedical Analysis
Volume 48, Issue 1, 10 September 2008, Pages 120-126
https://doi.org/10.1016/j.jpba.2008.05.018
4) Lines 100-101: What do the authors mean by methods for determination? Determination of what?
5) It is unlear from the background information provided that why did the authors performed this study? When a drug is approved by FDA such toxicity and metabolic studies are usually done. Are the authors saying that such studies, tocxicity and clearance, have not been conducted before? What is lacking on these drugs is not clear and what addition valuable information would be generated from the study in this manuscript it also unclear.
6) What is the purpose of the in silico analysis on these drugs? Are the binding sites for these drugs not known? Is is unknown that these drugs could be matabolised by liver? if all these are known, then could the authors please explain the value addition from their study.
