Tramadol Steady-State Pharmacokinetics of Immediate-Release Capsules and Sustained-Release Tablets in Dogs

Round 1

Reviewer 1 Report

This manuscript reports the results about the pharmacokinetic analysis and comparison of two formulations of tramadol administered in dogs. The objective of this study seems to be reasonable and has the potential to be applied in the clinical practise. However, the analysis of the data should be improved and several points must be addressed for the publication:

-       Abstract: it’s too short, result and discussion details should be improved in the abstract.

-       Drugs are not presented at the beginning. Their structure and main differences.

-       Tabla 1. Summarizes the validation information of LC-MS method for each substance, not the data about the analysis of the substances. That is in the supplementary material.

-       NCA. Measurements below LLOQ are not reliable, so they should be used for the analysis. Moreover, the use of a general value could end in misleading results.

-       Figure 1: values below LLOQ are not reliable. So they should not be included to calculate the measured values.

-       The terminal elimination half-life calculation should be more consistent for all data, so it could be done with the three last values.

Minor comments:

-       Add Title sections: Introduction, Materials and Methods (line 55), Results (line 90), Discussion (line 118), Conclusions (line 167).

-       Line 15: define abbreviations qid and sid in the abstract. And in all document

-       Line 15 and 16: How many samples are several? or How many serum concentrations were measured?

-       Title Supplement 2 is Figure S1, but there three figures inside, not just one. Title should be more general and each figure should be named as Figure S1, Figure S2 and Figure S3. Moreover, named figure A and B, instead of left and right, both in the image and in the footprint to understand easier.

-       Specific values from results are repeated along the discussion.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors,

congratulations to your great article.

I would just have one improvement suggestion.

Would it be possible to further elaborate on the accuracy of the measurements below the LLOQ?

It is stated that those values needed to be included in the calculation. However, please elaborate further on the impact.

Kind regards

Author Response

Please see attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors:

The Manuscript Tramadol steady-state pharmacokinetics of immediate-release capsules and sustained-release tablets in dogs is devoted to study the steady-state pharmacokinetics of a sustained-release tablet (SR) and immediate-release capsules (IR) in dogs comparing IR 50mg qid five times or SR 200mg sid twice.

Line 43 - 44: The authors cite "The analgesic efficacy of tramadol is the effect of both tramadol and the metabolite O-desmethyltramadol M1) [7]. M1 is reported to have a >200-fold higher affinity for the 44 μ-opioid receptor compared to tramadol. Because M1 is hardly produced in dogs, there are concerns about the analgesic efficacy of tramadol in this species [9-11]."

Although it is not provided background details on the metabolite production in dogs. What kind of enzymes would be responsible for the biotransformation? Is that similar to both sex (female and male dogs), as the investigation was done in female ones?

Regarding the LC-MS/MS analytical method, I think that below points are important to clarify the observed results:

 As mass detector is sensitive to both analyte and impurities. RT of 22.4 and 2.56 with the same m/z ratio is separated enough to give selectivity and confidence to quantification process?

About the method, was it fully validated? If so, give all the details about the detector used to quantify (number of quadrupoles, set up conditions) and describe reagents grade.

Please also show the validation parameters (sample clean up, detector conditions, precision, accuracy, robustness, etc) as suplemmentary material or, cite the reference, if previously published elsewhere.

-      line 73 Ammonium acetate; line 78 butabarbital as per Eur. Pharm.

-      line 74: Is that glacial acetic acid? there is no 100% acetic acid

-      lines 75-76 the concentration gradient went from 0-95% ACN in 2.5 min. How did such a short re-equilibration time impact on retention time of the analyte and internal standard? And over method robustness?

-      line 77-78: Please explain why both Cyanoimipramine and butobarbital were used as internal standards?

Lines 114-115 “ Because of great interindividual differences, the ranges in half-lives are also noted, besides the median.” How the authors explain such great variability?

Line 136-137 “It should, however, be remembered that the AUC might be over-estimated for SR formulations due to their flip-flop pharmacokinetics” How the authors can assure that sampling time or other phenomenon was not the reason for this “flip-flop” pharmacokinetics? Did you consider managing to avoid flip-flop pharmacokinetics and overestimated BA, with a longer duration of sampling? See doi: 10.1002/jps.24505; doi: 10.4155/tde.11.19

Line 159-160 “A limitation of this study is the high percentage of tramadol concentrations below LLOQ. Together with the small sample size of six dogs, this results in highly variable pharmacokinetic data.” Table 2 – Shows inference of values by LLOQ/2 (ie 2.5 μg/L) for 25% of samples from IR and 46% samples of SR tablets. Please explain the use of this putative value as the regulatory directions orientates to

According to EMEA guidelines on BA studies, AUC extrapolated from AUCt-last and infinity should be avoided and not correspond to more than 20% of the total AUC for good quality results. Although, data from table 2 shows AUC_inf/D % extrapolated for tramadol SR is 35.2% and, even higher for metabolites reaching up to 65.5% for M5. Please comment on EMEA statement.

Guidelines for the conduct of bioequivalence studies for veterinary medicinal products

Line 167- “We conclude that Tramagetic OD is a suitable formulation to reduce the dosing frequency of tramadol to once a day compared to four times daily with the IR capsules”. This statement does not seem to be supported by data shown herein. I would suggested rephrase it.

Author Response

Please see attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

A great improovement was seen for the manuscript, after the changes.