Structural Evolution of the Pharmaceutical Peptide Octreotide upon Controlled Relative Humidity and Temperature Variation
, Christina Papaefthymiou 1, Angelos Kontarinis 1, Maria Spiliopoulou 1, Dimitrios Koutoulas 1, Marios Konstantopoulos 1, Stamatina Kafetzi 1, Kleomenis Barlos 2, Kostas K. Barlos 2, Natalia Dadivanyan 3, Detlef Beckers 3, Thomas Degen 3, Andrew N. Fitch 4 and Irene Margiolaki 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for Authors2982221 Structural evolution of the pharmaceutical peptide octreotide….
Athanasiadou et al.
This paper reports an extensive study by PXRD of the crystal structure of octreotide as a function of changes in RH and T. The work has been carefully carried out as is generally well reported.
I have only a few specific comments that are laid out below, but my main ‘concern’ is over the driving force for the research reported, which comes across partly as an evaluation of the resilience of the compound to variable environment, partly as a demonstration of the capabilities of variable T/RH and partly as work that is ultimately relevant to the end use of the compound. This is an observation, not a criticism, but it does (to me) mean that the overall value of the paper to the community is somewhat limited, especially given that the work does not really fit naturally within the scope of the journal as it is described on the journal home page.
In terms of specific comments, these are:
Page 1, line 19: I can’t really see the evidence for enhanced ADME, so would be happier if the sentence stopped at “….development of a microcrystalline drug formulation.”
Page 1, line 3: Please italicise the ‘P’ of the space group symbol (search and apply throughout document as well).
Page 4, line 95: Any change to the physical form of the drug in the formulation is most likely to have an effect of the ‘A’ component of ‘ADME’….and as such I’d be inclined to restrict the potential to that component.
Page 4, line 118: There appears to be a font change in the references.
Page 6, Figure 2: I’m not sure that figure 2 really adds anything to the understanding of the paper; it could easily be removed.
Page 8, Figure 3: The quality of the plots in the figure is not good and in particular the insets are verging on the illegible. Whilst this may simply be down to resolution the proofs, I would still suggest that significantly larger plots should be used (e.g. a full page with two plots side-by-side, three rows of plots) to improve legibility and interpretation of the fits. Also, perhaps consider not using insets but rather a “lower panel” as you have done for figure 4?
Author Response
Dear Editor,
We are pleased to submit our revised manuscript entitled as:"Structural evolution of the pharmaceutical peptide octreotide upon controlled relative humidity and temperature variation” for consideration for publication in SynBio. We greatly appreciate the thoughtful feedback provided by the two reviewers and we have carefully addressed each of their comments in this revised version, which helped us to enhance the quality and the presentation of our manuscript.
Here is a detailed summary of the revisions made in response to the comments by Reviewer 1:
- “…my main ‘concern’ is over the driving force for the research reported, which comes across partly as an evaluation of the resilience of the compound to variable environment, partly as a demonstration of the capabilities of variable T/RH and partly as work that is ultimately relevant to the end use of the compound. This is an observation, not a criticism, but it does (to me) mean that the overall value of the paper to the community is somewhat limited, especially given that the work does not really fit naturally within the scope of the journal as it is described on the journal home page.”
Response: Thank you for your thoughtful assessment of our manuscript. We appreciate your insights into the perceived limitations of our research's applicability and value to the community. While our study focuses on octreotide, its implications extend beyond this specific compound. Peptide-based drugs constitute a vital class of pharmaceuticals and our investigation into octreotide's stability under varying conditions serves as a representative case study for this broader category.
Our findings, particularly regarding the remarkable stability of octreotide in its microcrystalline form, hold significant implications for peptide drug formulation. By demonstrating the feasibility and efficacy of microcrystalline therapeutic formulations, we present experimental data manifesting for the first time the enhanced stability of the microcrystalline octreotide. Moreover, considering octreotide's current availability solely as a suspension of amorphous counterparts, our research underscores the requirement of alternative formulations.
Through the application of advanced analytical techniques as X-ray powder diffraction (XRPD), our study not only offers valuable insights into octreotide's structural behavior but also provides practical implications for pharmaceutical formulation development and regulatory compliance. While we acknowledge that our study may not fit directly within the scope of the journal as described in the journal home page, we believe that our findings have broader impact for researchers and practitioners in pharmaceutical sciences and drug development. Given the mention of protein engineering and designed proteins in the journal's scope, we assert that our study contributes to the broader understanding of molecular stability and formulation strategies, thereby aligning with the overarching aims of the journal.
Finally, the following paragraph was added in the introduction section (page 3) in order to summarize the scope of this study. “While previous studies have investigated the stability of octreotide in sterile solution under controlled conditions, including storage at temperatures of 2-8°C for 24 months and elevated conditions of 25°C/60% rH (25), the in situ real-time study of the crystalline precipitate has not been previously reported. This presents an intriguing opportunity to explore the adaptation of octreotide in its crystalline form under varying environmental conditions.”
- “ Page 1, line 19: I can’t really see the evidence for enhanced ADME, so would be happier if the sentence stopped at “….development of a microcrystalline drug formulation.”
Response: Regarding the mention of enhanced ADME, we acknowledge your comment, but we are uncertain which specific sentence you are referring to. Probably it is on line 18 the mention of enhanced pharmacokinetics. However, we aim to clarify the significance of stability, particularly in the context of the microcrystalline form and its implications for pharmacokinetics. Stability is important in drug formulation, directly impacting pharmacokinetics. The stability of a formulation ensures consistent drug release, absorption kinetics and bioavailability, ultimately optimizing therapeutic outcomes. In the case of octreotide, the stability of its microcrystalline form offers several advantages. It provides consistent drug release, enhances bioavailability, and mitigates variability in pharmacokinetic parameters observed with amorphous formulations. This stability promotes more reliable and reproducible pharmacokinetic behavior, improving therapeutic outcomes and patient compliance. By demonstrating the stability of the microcrystalline form, we aim to contribute towards the development of more effective peptide-based medications. We hope this explanation clarifies the rationale behind our assertion regarding enhanced pharmacokinetics.
- “Page 1, line 3: Please italicise the ‘P’ of the space group symbol (search and apply throughout document as well).”
Response: Thank you for your attention to detail. We have made the necessary corrections to italicize the 'P' of the space group symbol throughout the manuscript, as per your suggestion. This modification has been applied consistently across the document to ensure uniform formatting.
- “ Page 4, line 95: Any change to the physical form of the drug in the formulation is most likely to have an effect of the ‘A’ component of ‘ADME’….and as such I’d be inclined to restrict the potential to that component.”
Response: Thank you for your insightful comment regarding the potential effects of changes in the physical form of the drug on the absorption component of ADME. We acknowledge the significance of absorption in the context of pharmaceutical formulations and agree that changes to the physical form are likely to have a primary impact on this aspect. While we recognize the importance of focusing on the absorption component, we believe it is essential to consider the broader impact on other components of ADME, as distribution, metabolism, and excretion. Changes in the physical form of the drug may influence not only its absorption but also its distribution within the body, metabolism by enzymatic processes, and subsequent excretion. Therefore, while our primary focus is on absorption, we acknowledge the interconnected nature of ADME processes and the potential for changes in the physical form to affect multiple aspects of a drug’s profile. We appreciate your suggestion to emphasize the 'A' component of ADME and we modified the text to ensure that our discussion appropriately addresses this aspect while also considering its broader implications on drug pharmacokinetics. The corresponding paragraph on page 17 has been revised as follows: “Given the potential impact of unexpected humidity fluctuations on pharmaceutical products, particularly on their absorption characteristics from their ADME profiles, the findings from rH experiments can provide valuable insights into product behavior and stability. These insights are essential for optimizing production and post-production processes to maintain the physicochemical and biochemical characteristics of drugs, with a primary focus on their absorption profiles within the body. By understanding how humidity variations affect the absorption of drugs, pharmaceutical companies can better design formulations to ensure consistent and predictable absorption kinetics, ultimately optimizing the drug's therapeutic efficacy.”
- “Page 4, line 118: There appears to be a font change in the references.”
Response: Thank you for bringing this to our attention. We have made revisions to ensure consistency, including numbering of the references as per the journal's guidelines. This adjustment not only addresses the font issue but also aligns with the recommended formatting for the references section.In addition, we revised all references provided in the manuscript and corrections were made where necessary.
- “ Page 6, Figure 2: I’m not sure that figure 2 really adds anything to the understanding of the paper; it could easily be removed.”
Response: We appreciate your observation regarding Figure 2. While it may not directly contribute to the understanding of the paper's main findings, we believe that it provides essential insights into the experimental configuration and setup. As such, we believe its inclusion aids in contextualizing the experimental methodology for readers, thereby enhancing the overall clarity and reproducibility of the experimental results.
- “ Page 8, Figure 3: The quality of the plots in the figure is not good and in particular the insets are verging on the illegible. Whilst this may simply be down to resolution the proofs, I would still suggest that significantly larger plots should be used (e.g. a full page with two plots side-by-side, three rows of plots) to improve legibility and interpretation of the fits. Also, perhaps consider not using insets but rather a “lower panel” as you have done for figure 4?”
Response: Thank you for your detailed feedback on Figure 3 and for your suggestions for improvement. Regarding the quality of the plots and the legibility of the insets, we have taken steps to enhance the clarity of all figures throughout the manuscript. Specifically, we have replaced all figures with versions at the highest resolution available from the outset, ensuring optimal image quality. This adjustment has been applied uniformly to all figures, including Figure 3. Additionally, we have reconsidered the layout of Figure 3 and have opted for a full-page with 3 rows and 2 plots side-by-side (no insets). We believe that these modifications have significantly enhanced the presentation of the data.
This manuscript has been submitted exclusively to SynBio.
Yours sincerely,
The authors:
Maria Athanasiadou, Christina Papaefthymiou, Angelos Kontarinis,
Maria Spiliopoulou, Dimitrios Koutoulas, Marios Konstantopoulos,
Stamatina Kafetzi, Kleomenis Barlos, Kostas K. Barlos,
Natalia Dadivanyan, Detlef Beckers, Thomas Degen, Andrew N. Fitch and
Irene Margiolaki
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe submitted manuscript provides for the XRPD evaluation of the synthetic peptide hormone octreotide after exposure to varying temperatures and humidity with the aim of validating the potential of using XRPD for the determination of structural degradation of this compound. This study is interesting and well described, with the results noting that octreotide is stable under the evaluated storage conditions. There are a few points for the authors to consider/expand on before this article can be suitable for publication. A list of these are as follows:
1. The importance of this study and its improvement on previous research should be expanded on in the Introduction.
2. XRPD provides a good understanding of the crystalline properties of molecules, however with regards to the model compound Octreotide, degradation of this peptide may not be determined using only just this method. The authors should expand on other analytical methods that could be used in conjunction with XRPD for the determination of peptide degradation.
3. The undertaken study was performed for a short period of time with a maximum temperature of 318.15K (45⁰C), which could influence the degradation potential of the molecule. As stability studies undertaken in accordance with ICH-compliant conditions usually occur over months, the time period used in this study and its possible influence on the stability of octreotide should be discussed.
4. The authors should also expand on any previous research that has focused on the temperature and relative humidity stability of octreotide, as well as on any studies that have analysed octreotide using XRPD.
5. The effect of peptide degradation on the therapeutic potential of octreotide should be provided to validate the importance of the results generated in this study.
6. Minor formatting: All abbreviations should be defined on first use.
Comments on the Quality of English LanguageThere are some minor grammatical errors in the manuscript. A proof-read of the manuscript is advised.
Author Response
Dear Editor,
We are pleased to submit our revised manuscript entitled as:"Structural evolution of the pharmaceutical peptide octreotide upon controlled relative humidity and temperature variation” for consideration for publication in SynBio. We greatly appreciate the thoughtful feedback provided by the two reviewers and we have carefully addressed each of their comments in this revised version, which helped us to enhance the quality and the presentation of our manuscript.
Here is a detailed summary of the revisions made in response to the comments by Reviewer 2:
- “ The importance of this study and its improvement on previous research should be expanded on in the Introduction.”
Response: Thank you for your report. The following paragraph was added in the introduction section (page 3) in order to summarize the scope of this study. “While previous studies have investigated the stability of octreotide in sterile solution under controlled conditions, including storage at temperatures of 2-8°C for 24 months and elevated conditions of 25°C/60% rH (25), the in situ real-time study of the crystalline precipitate has not been previously reported. This presents an intriguing opportunity to explore the adaptation of octreotide in its crystalline form under varying environmental conditions.”
In addition, we have revised the Introduction section to include a sub-section (1.4 Advancements and contributions of the current study) that explicitly discusses the importance of our study and its added value upon earlier studies, as suggested. We believe this addition enhances the clarity and significance of our research results.
- “XRPD provides a good understanding of the crystalline properties of molecules, however with regards to the model compound Octreotide, degradation of this peptide may not be determined using only just this method. The authors should expand on other analytical methods that could be used in conjunction with XRPD for the determination of peptide degradation.”
Response: Thank you for your insightful comment. We appreciate your recognition of the importance of employing complementary analytical methods to comprehensively assess peptide degradation. In response to your suggestion, we have integrated a paragraph on page 17 (Discussion section) on the limitations of XRPD in discerning molecular degradation mechanisms and proposed the incorporation of additional analytical techniques such as Fourier-transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). These methods offer complementary insights into molecular conformational changes, chemical stability and degradation products, thereby enhancing our understanding of peptide degradation pathways. The corresponding paragraph follows: “While XRPD serves as a robust method for monitoring the crystalline properties of peptides as octreotide, it is essential to acknowledge its limitations in discerning molecular degradation mechanisms comprehensively. To address this aspect, investigations usually integrate complementary analytical methods as Fourier-transform infrared spec-troscopy (FTIR) (56, 57), high-performance liquid chromatography (HPLC) (58) and mass spectrometry (MS) (59, 60), as previously utilized in peptide studies. These techniques offer complementary information on molecular conformational changes, chemical stability and degradation products, thereby enhancing our understanding of peptide stability and degradation pathways.
The duration and temperature range of our study were designed to provide insights into immediate structural responses of the microcrystalline specimen to humidity and temperature variations, simulating conditions relevant to accelerated stability testing. However, it is acknowledged that the condensed timeframe and maximum temperature of 318.15K (45°C) may not encompass the full spectrum of long-term stability assessments recommended by regulatory guide-lines. Future research may extend the present study duration to encompass longer-term stability evaluations, aligning more closely with ICH-compliant protocols (61). This extension would provide a comprehensive understanding of octreotide's stability profile over extended periods and address concerns regarding potential degradation influences associated with shorter study durations and elevated temperatures.
Finally, further experiments are currently ongoing to enhance the resolution limits of collected XRPD profiles and verify the reproducibility of observations. Towards this goal, the exceptionally significant improvement in the quality of synchrotron XRPD data fol-lowing the upgrade of ESRF to a fourth-generation circular accelerator, along with the utilization of a new data processing algorithm at beamline ID22, creates new expectations for more efficient analysis of XRPD diffraction data and for more precise structure de-termination with enhanced atomic resolution (39). These measurements will contribute to the identification of optimal dehydration protocols and provide a deeper understanding of the structural changes induced by humidity variations, ultimately advancing phar-maceutical research and development efforts.
In summary, the experimental approach presented herein lays the groundwork for standardizing investigations into structural changes of primary polycrystalline samples under varying humidity and temperature conditions, offering insights into crucial bio-logical phenomena.”
- “The undertaken study was performed for a short period of time with a maximum temperature of 318.15K (45⁰C), which could influence the degradation potential of the molecule. As stability studies undertaken in accordance with ICH-compliant conditions usually occur over months, the time period used in this study and its possible influence on the stability of octreotide should be discussed.”
Response: We acknowledge the relatively short duration and temperature variation presented herein. While our focus was on immediate structural responses to environmental fluctuations, we recognize the importance of longer-term stability evaluations. We now consider extending the study’s duration in future research to align with ICH-compliant protocols and provide a more comprehensive understanding of octreotide's stability profile. We've addressed your comment by acknowledging the study's short duration and temperature range in a new paragraph added to the discussion section (page 17- new paragraph follows). “The duration and temperature range of our study were designed to provide insights into immediate structural responses of the microcrystalline specimen to humidity and temperature variations, simulating conditions relevant to accelerated stability testing. However, it is acknowledged that the condensed timeframe and maximum temperature of 318.15K (45°C) may not encompass the full spectrum of long-term stability assessments recommended by regulatory guidelines. Future research may extend the present study duration to encompass longer-term stability evaluations, aligning more closely with ICH-compliant protocols (61). This extension would provide a comprehensive un-derstanding of octreotide's stability profile over extended periods and address concerns regarding potential degradation influences associated with shorter study durations and elevated temperatures.”
- “ The authors should also expand on any previous research that has focused on the temperature and relative humidity stability of octreotide, as well as on any studies that have analysed octreotide using XRPD.”
- “The effect of peptide degradation on the therapeutic potential of octreotide should be provided to validate the importance of the results generated in this study.”
Response: Thank you for your suggestions. We have taken your suggestions into account and have already incorporated a discussion on previous research regarding octreotide's stability under various conditions, as well as studies utilizing XRPD analysis, in the revised introduction and discussion sections as these are provided above. We believe this addition provides a more comprehensive context for our study. Further details would be beyond the scope of this study.
- “Minor formatting: All abbreviations should be defined on first use.”
Response: Thank you for bringing this to our attention. We defined all abbreviations upon their first use in the manuscript.
- “There are some minor grammatical errors in the manuscript. A proof-read of the manuscript is advised.”
Response: Thank you for your feedback. We appreciate your attention to detail and we conducted a thorough proofreading of the manuscript to address any minor grammatical errors.
This manuscript has been submitted exclusively to SynBio.
Yours sincerely,
The authors:
Maria Athanasiadou, Christina Papaefthymiou, Angelos Kontarinis,
Maria Spiliopoulou, Dimitrios Koutoulas, Marios Konstantopoulos,
Stamatina Kafetzi, Kleomenis Barlos, Kostas K. Barlos,
Natalia Dadivanyan, Detlef Beckers, Thomas Degen, Andrew N. Fitch and
Irene Margiolaki
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have adequately addressed all comments provided previously. This manuscript may therefore be accepted for publication.
