Clodronate Reduces ATP-Containing Microvesicle Releasing Induced by Nociceptive Stimuli in Human Keratinocytes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The abstract is comprehensive, but it could be more concise. The introduction provides a good background but needs a clearer flow from general information about bisphosphonates to the specific study. Streamline the abstract to focus on key findings and relevance. In the introduction, clearly state the research question and the rationale for using Clodronate in the context of nociceptive stimuli.

 

2. The paper uses capsaicin and KOH as nociceptive stimuli but lacks a detailed justification for their selection and their relevance to human conditions. Provide more background on why capsaicin and KOH are appropriate models for studying nociceptive responses in keratinocytes and their relevance to clinical pain conditions.

 

3. The control groups used for comparison should be explicitly described in the methods section to clarify the baseline against which the effects of nociceptive stimuli and Clodronate are measured. Ensure that control conditions are clearly defined and include untreated keratinocytes, keratinocytes treated with nociceptive stimuli alone, and keratinocytes treated with Clodronate alone.

 

4. The paper mentions using ANOVA and Bonferroni’s post hoc tests but lacks detail on the specific statistical tests applied to each set of data. Provide a detailed description of the statistical analysis performed for each experiment, including assumptions checked and rationale for choosing specific tests.

 

5. The mechanism by which Clodronate reduces MVs release is discussed but not experimentally verified. Include experiments to directly measure the uptake of Clodronate in keratinocytes and its interaction with VNUT or other relevant cellular targets to confirm the proposed mechanism.

 

6. The role of Dansylcadaverine in inhibiting Clodronate uptake and its partial effects need further clarification. Perform additional experiments to determine the exact impact of Dansylcadaverine on Clodronate uptake and MVs release. This could involve varying concentrations of Dansylcadaverine and measuring Clodronate uptake directly.

 

7. The methodology for quantifying ATP in MVs using luciferin-luciferase reaction is briefly mentioned but lacks detail. Provide a detailed protocol for ATP quantification, including sample preparation, controls for ATP measurement, and calibration of the luminescence signal.

 

8. The potential clinical application of Clodronate as a topical analgesic is mentioned but not sufficiently discussed. Expand on the potential clinical implications of the findings, including any preliminary data on the topical application of Clodronate and future research directions needed to translate these findings to clinical use.

 

9. The study suggests further investigation into the relationship between VNUT and MVs shedding but does not propose specific experiments. Propose specific follow-up experiments to explore the role of VNUT in MVs shedding, such as using VNUT knockdown or overexpression models, and evaluating the impact on ATP release and nociception.

 

10. Perform a detailed dose-response analysis of Clodronate and nociceptive stimuli to identify the optimal concentrations that affect MVs release and ATP content.

 

11. Include in vivo experiments to validate the findings in an animal model of nociception, ensuring that the observed effects translate beyond in vitro conditions.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

 Reviewer 1

The authors thank the reviewer for the kind indications for paper improvement

1. The abstract is comprehensive, but it could be more concise. The introduction provides a good background but needs a clearer flow from general information about bisphosphonates to the specific study. Streamline the abstract to focus on key findings and relevance. In the introduction, clearly state the research question and the rationale for using Clodronate in the context of nociceptive stimuli.

The authors tried to write an abstract as concise as possible using the 200 words as indicated by the journal and taking into account the complexity of the scientific hypothesis.

 

2. The paper uses capsaicin and KOH as nociceptive stimuli but lacks a detailed justification for their selection and their relevance to human conditions. Provide more background on why capsaicin and KOH are appropriate models for studying nociceptive responses in keratinocytes and their relevance to clinical pain conditions.

 

Both Capsaicin and KOH use justification ais present in the discussion section (line 201-211). Moreover some additional information and a new bibliograpihic sentence has been added for capsaicin as requested by another reviewer.

 

3. The control groups used for comparison should be explicitly described in the methods section to clarify the baseline against which the effects of nociceptive stimuli and Clodronate are measured. Ensure that control conditions are clearly defined and include untreated keratinocytes, keratinocytes treated with nociceptive stimuli alone, and keratinocytes treated with Clodronate alone.

 

The control groups have been indicated for every test performed in the M&M section. In particular pag 9 line 278 for MTT test, pag 10 line 298 for MVs release and ATP measuremen (pg 10 line 339).

 

4. The paper mentions using ANOVA and Bonferroni’s post hoc tests but lacks detail on the specific statistical tests applied to each set of data. Provide a detailed description of the statistical analysis performed for each experiment, including assumptions checked and rationale for choosing specific tests.

 

ANOVA test (one way) allows you to compare the mean values ​​of a given effect in any number of groups. For example the toxicity in cells treated with clodronate or capsaicin or KOH compared to untreated cells.

The Bonferroni test (post hoc ad anova) is used to compare all the means with all the others as the conditions increase and to be much more sure that the differences observed are real and significant.

In our experience we have never been asked to explain this in the statistics section of the M&M section

 

5. The mechanism by which Clodronate reduces MVs release is discussed but not experimentally verified. Include experiments to directly measure the uptake of Clodronate in keratinocytes and its interaction with VNUT or other relevant cellular targets to confirm the proposed mechanism.

 

This preliminary study is centered on the effect of clodronate on MVs release as a possible analgesic effect.  We are working on the mechanism that is about to be submitted in another in vitro paper.

We mentioned this inn the Discussion section

 

6. The role of Dansylcadaverine in inhibiting Clodronate uptake and its partial effects need further clarification. Perform additional experiments to determine the exact impact of Dansylcadaverine on Clodronate uptake and MVs release. This could involve varying concentrations of Dansylcadaverine and measuring Clodronate uptake directly.

 

We add information about this in the Discussion section as a part of the paper limits.

 

7. The methodology for quantifying ATP in MVs using luciferin-luciferase reaction is briefly mentioned but lacks detail. Provide a detailed protocol for ATP quantification, including sample preparation, controls for ATP measurement, and calibration of the luminescence signal.

 

A more detailed description of the ATP method has been added to the M&M section.

 

 

8. The potential clinical application of Clodronate as a topical analgesic is mentioned but not sufficiently discussed. Expand on the potential clinical implications of the findings, including any preliminary data on the topical application of Clodronate and future research directions needed to translate these findings to clinical use.

 

We add a short part about this in the conclusion section.

 

 

9. The study suggests further investigation into the relationship between VNUT and MVs shedding but does not propose specific experiments. Propose specific follow-up experiments to explore the role of VNUT in MVs shedding, such as using VNUT knockdown or overexpression models, and evaluating the impact on ATP release and nociception.

 

We add this indication in the discussion section.

 

10. Perform a detailed dose-response analysis of Clodronate and nociceptive stimuli to identify the optimal concentrations that affect MVs release and ATP content.

 

This is a part of work limitations that we add to the discussion section, However we consider this an initial and preliminary study.

11. Include in vivo experiments to validate the findings in an animal model of nociception, ensuring that the observed effects translate beyond in vitro conditions.

We are actually working on an in vivo study using rats treated topically with capsaicin and will use a modified form of Clodronate to be used before or after capsaicin treatment to measure the animal's reaction which we can interpret as pain. We prefer not to comment on this as it is an ongoing project.

 

 

 

 

 

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

This is a report from a research work on the Clodronate (Clod), which is a first-generation bisphosphonate. Cold acts as a natural analgesic inhibiting vesicular storage of the nociception mediator ATP by vesicular nucleotide transporter (VNUT). Epidermal keratinocytes are involved in cutaneous nociception by accumulating ATP within vesicles which are released following different stimulations. Under stress condition keratinocyte produce microvesicles (MVs) that their secretion has been identified as a novel and universal mode of intercellular communication between cells.

Based on this background, the authors aimed to evaluate if two nociceptive stimuli (Capsicin and KOH) could stimulate MVs shedding from human keratinocytes if these MVs could contain ATP, and if Clod could inhibit this shedding process. To address this, the authors employed a cellular model, HaCaT keratinocyte monolayer, and used stimulators (both Capsaicin and KOH) to provoke MVs release. The findings demonstrated that after 3 hours of incubation, MVs were released following the application of both stimulators and the released MVs contained ATP. Clod (5μM) reduced Capsaicin-induced MVs release and KOH-induced MVs release. The authors proposed – based on the findings – that Clod could be a pharmacological basis for developing new local analgesic drugs.

The authors are encouraged to address these points in the revised version of their manuscript:

1.       The authors have indicated that bisphosphonates can act as analgesics in the treatment of pain associated with bone metastases. Please expand on this and elaborate on the mechanism of action.

2.       Please add more information on the role of keratinocytes in cutaneous nociception. Can they separately act or do they need collaboration with cutaneous nerves to signal nociception?

3.       Please justify the use of stimulators such as capsaicin. Capsaicin has been used to subside pain as its patch is used in neuropathic pain. Has this substance dual action? Pain induction and pain reduction? Please elaborate to avoid confusion for readers.

4.       What is KOH? Add the abbreviation full term first-time use in the article. In general, make sure that all abbreviations have been defined. For example in Fig 2 legend, define DC, when you use the compound name.

5.       Please justify the concentrations used for Cold. Are those two based on pilot studies of own or from existing literature? Add the information, please.

6.       Add the process of ATP luminescence measurement that has been presented in Figure 3. What is the accuracy of this method and the range of acceptable error?

7.       The experimental procedure here is an in vitro test. How do the authors interpret the translational potential of in vivo models? What are the risks and potentials? Please elaborate and add.

8.       Please add the limitations of this study and sources of bias.

9.       If hypothetically it comes to the application as analgesics in vivo, shall it be by topical skin application or any other routes of administration? Please explain.

1.   Do the authors speculate a dose-response effect? The higher the dose, the higher the analgesic effect, or would that be a ceiling effect before toxicity? Please elaborate and add.

1.   What will be the potential side effects of a bisphosphonate to be used against pain? Can it be justified in front of other safer drugs for his purpose? Please elaborate and add.

1.   The authors have concluded that this study can be base for the development of local analgesics following the findings. How the authors would ensure that the effect is local? What would be the potential for systemic take and circulation after local application and the harmful consequences?

 

Author Response

Reviewer 2

The authors thank the reviewer for the kind indications for paper improvement

 

1. The authors have indicated that bisphosphonates can act as analgesics in the treatment of pain associated with bone metastases. Please expand on this and elaborate on the mechanism of action.

 

Analgesic effects of clodronate and its possible mechanism has been described in Introduction  (line line 75-78).

 

2. Please add more information on the role of keratinocytes in cutaneous nociception. Can they separately act or do they need collaboration with cutaneous nerves to signal nociception?

 

The role of keratinocyte in nociception has been described in line 79 to 89 at page 3 (introduction section)

 

3. Please justify the use of stimulators such as capsaicin. Capsaicin has been used to subside pain as its patch is used in neuropathic pain. Has this substance dual action? Pain induction and pain reduction? Please elaborate to avoid confusion for readers.

 

Two sentences have been added in the Discussion section together with a new bibliographic entry [Arora et al., 2020,  new n.26] to explain the dual activity of capsaicin (low and high concentrations)

 

4. What is KOH? Add the abbreviation full term first-time use in the article. In general, make sure that all abbreviations have been defined. For example in Fig 2 legend, define DC, when you use the compound name.

We added KOH explanation and modified Fig,2 legend

 

5. Please justify the concentrations used for Cold. Are those two based on pilot studies of own or from existing literature? Add the information, please.

Clodronate analgesic activity has been correlated to its inhibiting activity toward VNUT in neurons. The Clodronate IC 50 for VNUT is 0,0156 µM (ref. n.34) and it has been used in in vitro studies at concentration of 30 or 1000 µM (ref. n. 36) therefore we decided to use a reasonable range of concentration in this first study on keratinocyte between 0.05 to 5 µM to be sure not to create eventual toxicity. We add a phrase in M&M section to justify Clodronate concentration range.

 

 

6. Add the process of ATP luminescence measurement that has been presented in Figure 3. What is the accuracy of this method and the range of acceptable error?

As indicated by the producer of the kit used for ATP measure, the method used can  detect as little as a single cell or 0.01 picomole of ATP. The signal produced is linear within 6 orders of magnitude.

 

7. The experimental procedure here is an in vitro test. How do the authors interpret the translational potential of in vivo models? What are the risks and potentials? Please elaborate and add.

 

We are actually working on an in vivo study using rats treated topically with capsaicin and will use a modified form of Clodronate to be used before or after capsaicin treatment to measure the animal's reaction which we can interpret as pain. We prefer not to comment on this as it is an ongoing project.

 

8. Please add the limitations of this study and sources of bias.

We add a part about work limitation in the Discussion section.

 

9.If hypothetically it comes to the application as analgesics in vivo, shall it be by topical skin application or any other routes of administration? Please explain.

We add  a part about this in the conclusion section

 

10. Do the authors speculate a dose-response effect? The higher the dose, the higher the analgesic effect, or would that be a ceiling effect before toxicity? Please elaborate and add.

We did not have collect data on the dose-response effect on MV release as we preferred to use the highest non-toxic Clodronate concentration tested (5 µM). However, as shown in Fig.3, there is a clear dose-response effect of Clodronate on the ATP concentration.

 

11. What will be the potential side effects of a bisphosphonate to be used against pain? Can it be justified in front of other safer drugs for his purpose? Please elaborate and add.

 

The most important side effect of oral or intravenous administration of bisphosphonates in patients suffering from cancer or osteoporosis is osteonecrosis of the mandible and/or maxilla, generally associated with tooth extraction and/or local infection (including osteomyelitis). Many of these patients were also treated with chemotherapy and corticosteroids. Normally a patient with intravenous clodronate is administered at a concentration of 100 mg per week for prolonged periods (years) (Filipponi et al., 1996) . In our experimentation for topical use of clodronate drug concentrations and treatment periods will be enormously lower than those that involve toxicity towards the patient.

Filipponi P, Cristallini S, Rizzello E, et al. Cyclical intravenous clodronate in postmenopausal osteoporosis: Results of a long-term clinical trial. Bone 1996;18:179-84.

 

• The authors have concluded that this study can be base for the development of local analgesics following the findings. How the authors would ensure that the effect is local? What would be the potential for systemic take and circulation after local application and the harmful consequences?

The question posed by the reviewer concerns all drugs used topically, so systemic diffusion cannot be ruled out. However, there are two limits to the diffusion of clodronate at the systemic level: clodronate does not diffuse easily and is not easily uptaken by cells due to its chemical conformation and the relatively low concentration (between 5 and 50 µM) to be used in a study in vivo.

This is one of the reasons why we are developing a clodronate formulation optimized for topical administration and covered by patent.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have no more comments.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed the points and revised the manuscript accordingly. Thanks. There are no further comments.