<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT04473976</url>
  </required_header>
  <id_info>
    <org_study_id>CGRP-microRNA2020</org_study_id>
    <nct_id>NCT04473976</nct_id>
  </id_info>
  <brief_title>Plasma Levels of CGRP and Expression of Specific microRNAs in Blood Cells of Episodic and Chronic Migraine Subjects</brief_title>
  <acronym>CGRP-RNA</acronym>
  <official_title>Plasma Levels of CGRP and Expression of Specific microRNAs in Blood Cells of Episodic and Chronic Migraine Subjects: Toward the Identification of a Panel of Peripheral Biomarkers of Migraine?</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>IRCCS National Neurological Institute &quot;C. Mondino&quot; Foundation</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
    <collaborator>
      <agency>University of Pavia</agency>
      <agency_class>Other</agency_class>
    </collaborator>
  </sponsors>
  <source>IRCCS National Neurological Institute &quot;C. Mondino&quot; Foundation</source>
  <oversight_info>
    <has_dmc>No</has_dmc>
    <is_fda_regulated_drug>No</is_fda_regulated_drug>
    <is_fda_regulated_device>No</is_fda_regulated_device>
  </oversight_info>
  <brief_summary>
    <textblock>
      Migraine can manifest with an episodic or chronic pattern in a continuum of disease severity.&#xD;
      Multiple factors are associated to the transformation of the pattern form episodic to&#xD;
      chronic. Of these, the most consistently reported is the overuse of medications (MO) for the&#xD;
      acute treatment of attacks. Knowledge of the mechanisms through which MO facilitates the&#xD;
      transformation of episodic migraine (EM) into chronic migraine (CM) is very limited. In order&#xD;
      insights into these mechanisms, the present study was aimed at identifying possible&#xD;
      peripheral biomarkers associated to the 2 forms of migraine and to the presence of MO.&#xD;
&#xD;
      The investigators evaluated CGRP plasma levels and the expression of miR-34a-5p and&#xD;
      miR-382-5p in peripheral blood mononuclear cells of subjects with episodic migraine (EM,&#xD;
      n=27) and CM-MO (n=28). CM-MO group was also tested 2 months after an in-hospital&#xD;
      detoxification protocol.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      Chronic migraine (CM) is a highly disabling condition that frequently manifests as a negative&#xD;
      evolution of episodic migraine (EM) taking place over years. In a recent meta-analysis on the&#xD;
      predictors of migraine chronification, a number of monthly headache days &gt;10 showed the&#xD;
      strongest level of evidence, with depression and low household income being supported by&#xD;
      moderate evidence. In the same meta-analysis, medication overuse (MO) was associated to the&#xD;
      highest risk ratio in a random-effects model (RR 8.82; 95% Confidence Interval (CI), 2.88-27)&#xD;
      but the strength of evidence was rated 'very low'due to substantial heterogeneity among&#xD;
      studies.&#xD;
&#xD;
      It is likely that CM is the result of the dynamic interaction of multiple co-factors acting&#xD;
      on a substrate represented by a more aggressive type of migraine. This hypothesis would&#xD;
      explain why as many as 75% of CM subjects can spontaneously fluctuate around chronicity and&#xD;
      episodicity. On the other hand, multiple pieces of evidence show that withdrawal from overuse&#xD;
      medications induces a clinically meaningful improvement in a large percentage of subjects,&#xD;
      with rates of benefit that are too high to be ascribed to a simple placebo effect.&#xD;
&#xD;
      The mechanisms underlying the negative effect of MO in migraine outcome are largely unknown.&#xD;
      Neural mechanisms including a combination of nociceptive facilitation with weakened&#xD;
      descending pain inhibition, associated with trigeminal hyperexcitability, may be involved.&#xD;
      The loss of diffuse descending inhibition has been demonstrated in a preclinical model of CM&#xD;
      with MO. In this frame, it is worth noting that the investigators have recently reported a&#xD;
      marked derangement of the endocannabinoid system in subjects with CM and MO (CM-MO), which&#xD;
      was more marked when compared with subjects suffering from episodic migraine (EM).&#xD;
      Interestingly, detoxification induced in CM-MO subjects the normalization of pain thresholds&#xD;
      paralleled by the reduction in headache frequency. Pre-clinical studies show that triptans,&#xD;
      the specific symptomatic migraine drugs, induce a condition of hyperalgesia when administered&#xD;
      chronically. Thus, it is possible that acute migraine medications taken in repeated episodes&#xD;
      of pain may amplify the consequences of nociceptor activation and increase the probability of&#xD;
      subsequent migraine attacks, together with the risk of medication overuse.&#xD;
&#xD;
      In this context of multiple concurrent causes, it seems extremely important to investigate in&#xD;
      more depth the mechanisms that may be involved in CM and MO. Calcitonin gene-related peptide&#xD;
      (CGRP) undoubtedly plays an important, though not exclusive, role in the generation of&#xD;
      migraine headaches. CGRP receptors are localized in the sites involved in migraine&#xD;
      pathogenesis. CGRP is involved in mast cell degranulation, neurogenic inflammation, and&#xD;
      vasodilation. It has been shown that CGRP induces InterLeukin-6 (IL-6) gene expression in&#xD;
      macrophages by upregulation of circular RNA_007893, a modulator of MicroRNA-485-5p. MicroRNAs&#xD;
      (miRNAs) are involved in the generation and maintenance of chronic pain and several lines of&#xD;
      evidence suggest that specific miRNAs may play a role in migraine pain. In a previous&#xD;
      clinical study, Andersen and colleagues found an increased expression of miR-34a-5p during&#xD;
      migraine attacks, while miR-382-5p levels increased also in the attack-free phase. In&#xD;
      addition, the peripheral expression of miR-34a-5p decreased in the saliva of young&#xD;
      migraineurs patients under drug treatment, thus suggesting a possible role in the prediction&#xD;
      of therapeutic response. At present, no reliable individual biomarker of migraine and its&#xD;
      subtypes has been identified, though multiple molecules have been proposed and supported by&#xD;
      promising results.&#xD;
&#xD;
      In this study, the investigators assayed the plasma levels of CGRP and the expression of&#xD;
      miRNAs in PBMCs of patients with EM and CM-MO in order to identify individual or a panel of&#xD;
      potential biomarkers of migraine subtypes. As secondary outcome, the investigators evaluated&#xD;
      the changes in CGRP and miRNAs levels after detoxification in the subjects with CM-MO in&#xD;
      order to gather more insights into the mechanisms that are involved in the improvement of&#xD;
      migraine pattern following the withdrawal of the overused medications.&#xD;
&#xD;
      This is a cross-sectional observational controlled study with 2 groups (EM and CM-MO),&#xD;
      integrated with a prospective open label interventional trial to assess the effect of&#xD;
      detoxification in the CM-MO group on the biomarkers of interest. Samples were labeled with&#xD;
      numerical codes and all biochemical determinations were performed by researchers who were&#xD;
      blind to the diagnosis.&#xD;
&#xD;
      At baseline (Day 0), all patients underwent a visit with a neurologist of the Headache&#xD;
      Science Centre during which clinical/demographical data were recorded and inclusion/exclusion&#xD;
      criteria were checked. If the criteria were met, subjects underwent peripheral venipuncture&#xD;
      for the evaluation of CGRP, miR-382-5p, and miR-34a-5p levels.&#xD;
&#xD;
      Patients with CM-MO were hospitalized for a 7-day standardized detoxification protocol,&#xD;
      consisting in abrupt withdrawal of overused drugs associated to intravenous therapy twice&#xD;
      daily (08:00 a.m. and 4:00 p.m.) with isotonic 0.9% Sodium Chloride (NaCl) saline 500 ml +&#xD;
      cyanocobalamin 2500 mcg + folic acid 0.70 mg + nicotinamide 12 mg + ascorbic acid 150 mg +&#xD;
      sodic glutathione 600 mg + delorazepam 0.5 mg. Two months after hospital discharge (Day 1),&#xD;
      the CM-MO patients returned for a follow-up visit, during which clinical data were recorded&#xD;
      and a second venous blood sample obtained from their ante-cubital vein.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Completed</overall_status>
  <start_date type="Actual">December 15, 2017</start_date>
  <completion_date type="Actual">June 15, 2019</completion_date>
  <primary_completion_date type="Actual">April 15, 2019</primary_completion_date>
  <study_type>Observational</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <observational_model>Other</observational_model>
    <time_perspective>Cross-Sectional</time_perspective>
  </study_design_info>
  <primary_outcome>
    <measure>Plasma levels of CGRP</measure>
    <time_frame>Day 0 at the enrolment</time_frame>
    <description>In order to identify individual or a panel of potential biomarkers of migraine subtypes</description>
  </primary_outcome>
  <primary_outcome>
    <measure>Expression of miRNAs in PBMCs</measure>
    <time_frame>Day 0 at the enrolment</time_frame>
    <description>In order to identify individual or a panel of potential biomarkers of migraine subtypes</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>Changes in CGRP levels</measure>
    <time_frame>Day1 - Two months after Day 0</time_frame>
    <description>In the subjects with CM-MO after detoxification in order to gather more insights into the mechanisms that are involved in the improvement of migraine pattern following the withdrawal of the overused medications.</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Changes in miRNAs levels</measure>
    <time_frame>Day 1 - Two months after Day 0</time_frame>
    <description>In the subjects with CM-MO after detoxification in order to gather more insights into the mechanisms that are involved in the improvement of migraine pattern following the withdrawal of the overused medications.</description>
  </secondary_outcome>
  <enrollment type="Actual">55</enrollment>
  <condition>Migraine</condition>
  <biospec_retention>Samples Without DNA</biospec_retention>
  <biospec_descr>
    <textblock>
      Blood samples from the cubital vein of each patient was put into a 5 ml lavender tube. Plasma&#xD;
      samples were prepared by centrifugation and then stored at -80°C. Plasma CGRP levels were&#xD;
      measured using a commercial enzyme-linked immuno-sorbent assay kit.&#xD;
&#xD;
      The miRNAs expression was evaluated by real-time reverse transcription (RT) Polymerase Chain&#xD;
      Reaction (PCR) in peripheral blood mononuclear cell (PBMCs). PBMCs were isolated immediately&#xD;
      after blood collection. Blood samples were collected within ethylenediamine tetra-acetic acid&#xD;
      (EDTA) containing tube and diluted in 1:1 ratio with Phosphate Buffer Saline (PBS). Then,&#xD;
      diluted blood samples were slowly loaded into Ficoll separating solution (15 ml) (Sigma) and&#xD;
      centrifuged at 800 g for 30 min at room temperature. PBMCs accumulated as the middle white&#xD;
      monolayer, were washed twice in sterile PBS at 300 g for 15 min. After washing, PBMCs pellet&#xD;
      was resuspended in trizol and stored at -80 °C until use.&#xD;
    </textblock>
  </biospec_descr>
  <eligibility>
    <study_pop>
      <textblock>
        Twenty-seven subjects with EM and 28 subjects with CM-MO were consecutively enrolled among&#xD;
        patients attending the outpatient clinics of the Headache Science Centre of the IRCCS&#xD;
        Mondino Foundation of Pavia (Italy).&#xD;
      </textblock>
    </study_pop>
    <sampling_method>Non-Probability Sample</sampling_method>
    <criteria>
      <textblock>
        Inclusion Criteria for EM subjects:&#xD;
&#xD;
          -  diagnosis of migraine without aura according to The International Classification&#xD;
             Headache Disorders 3rd edition (ICHD-3) criteria;&#xD;
&#xD;
          -  documented history of EM for at least 10 years before enrollment;&#xD;
&#xD;
          -  negative life-time history of CM.&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  headache or headache with migraine features and mild intensity (less than 4 on 0 to 10&#xD;
             visual analogue scale) in the 24 hours before blood sampling;&#xD;
&#xD;
          -  intake of acute anti-migraine medications in the 24 hours before blood sampling.&#xD;
&#xD;
        Inclusion criteria for CM-MO patients:&#xD;
&#xD;
          -  diagnosis of CM and MO according to ICHD-3 criteria;&#xD;
&#xD;
          -  documented pattern of stable CM in the 5 years prior to enrollment, without any&#xD;
             remission period. This latter point was verified with a high degree of confidence&#xD;
             combining the information obtained from the patients' history with their medical&#xD;
             records, including their headache diaries.&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  headache or headache with migraine features and mild intensity (less than 4 on 0 to 10&#xD;
             visual analogue scale) in the 24 hours before blood sampling;&#xD;
&#xD;
          -  intake of acute anti-migraine medications in the 24 hours before blood sampling.&#xD;
      </textblock>
    </criteria>
    <gender>All</gender>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>N/A</maximum_age>
    <healthy_volunteers>No</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>Cristina Tassorelli, Prof</last_name>
    <role>Principal Investigator</role>
    <affiliation>IRCCS Mondino Foundation, Pavia</affiliation>
  </overall_official>
  <location>
    <facility>
      <name>Headache Science Center</name>
      <address>
        <city>Pavia</city>
        <zip>27100</zip>
        <country>Italy</country>
      </address>
    </facility>
  </location>
  <location_countries>
    <country>Italy</country>
  </location_countries>
  <reference>
    <citation>Diener HC, Tassorelli C, Dodick DW, Silberstein SD, Lipton RB, Ashina M, Becker WJ, Ferrari MD, Goadsby PJ, Pozo-Rosich P, Wang SJ, Mandrekar J; International Headache Society Clinical Trials Standing Committee. Guidelines of the International Headache Society for controlled trials of acute treatment of migraine attacks in adults: Fourth edition. Cephalalgia. 2019 May;39(6):687-710. doi: 10.1177/0333102419828967. Epub 2019 Feb 26.</citation>
    <PMID>30806518</PMID>
  </reference>
  <reference>
    <citation>Xu J, Kong F, Buse DC. Predictors of episodic migraine transformation to chronic migraine: A systematic review and meta-analysis of observational cohort studies. Cephalalgia. 2020 Apr;40(5):503-516. doi: 10.1177/0333102419883355. Epub 2019 Oct 21.</citation>
    <PMID>31635478</PMID>
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  <reference>
    <citation>Serrano D, Lipton RB, Scher AI, Reed ML, Stewart WBF, Adams AM, Buse DC. Fluctuations in episodic and chronic migraine status over the course of 1 year: implications for diagnosis, treatment and clinical trial design. J Headache Pain. 2017 Oct 4;18(1):101. doi: 10.1186/s10194-017-0787-1.</citation>
    <PMID>28980171</PMID>
  </reference>
  <reference>
    <citation>Tassorelli C, Jensen R, Allena M, De Icco R, Sances G, Katsarava Z, Lainez M, Leston J, Fadic R, Spadafora S, Pagani M, Nappi G; the COMOESTAS Consortium. A consensus protocol for the management of medication-overuse headache: Evaluation in a multicentric, multinational study. Cephalalgia. 2014 Aug;34(9):645-655. doi: 10.1177/0333102414521508. Epub 2014 Feb 20.</citation>
    <PMID>24558185</PMID>
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  <reference>
    <citation>Rossi P, Faroni JV, Tassorelli C, Nappi G. Advice alone versus structured detoxification programmes for complicated medication overuse headache (MOH): a prospective, randomized, open-label trial. J Headache Pain. 2013 Feb 8;14(1):10. doi: 10.1186/1129-2377-14-10.</citation>
    <PMID>23565591</PMID>
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    <PMID>31174999</PMID>
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    <citation>De Felice M, Ossipov MH, Porreca F. Persistent medication-induced neural adaptations, descending facilitation, and medication overuse headache. Curr Opin Neurol. 2011 Jun;24(3):193-6. doi: 10.1097/WCO.0b013e328346af25.</citation>
    <PMID>21467931</PMID>
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  <reference>
    <citation>Munksgaard SB, Bendtsen L, Jensen RH. Modulation of central sensitisation by detoxification in MOH: results of a 12-month detoxification study. Cephalalgia. 2013 May;33(7):444-53. doi: 10.1177/0333102412475235. Epub 2013 Feb 21.</citation>
    <PMID>23431023</PMID>
  </reference>
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    <citation>De Felice M, Ossipov MH, Wang R, Lai J, Chichorro J, Meng I, Dodick DW, Vanderah TW, Dussor G, Porreca F. Triptan-induced latent sensitization: a possible basis for medication overuse headache. Ann Neurol. 2010 Mar;67(3):325-37. doi: 10.1002/ana.21897.</citation>
    <PMID>20373344</PMID>
  </reference>
  <reference>
    <citation>Lambru G, Andreou AP, Guglielmetti M, Martelletti P. Emerging drugs for migraine treatment: an update. Expert Opin Emerg Drugs. 2018 Dec;23(4):301-318. doi: 10.1080/14728214.2018.1552939. Epub 2018 Nov 30.</citation>
    <PMID>30484333</PMID>
  </reference>
  <reference>
    <citation>Deng T, Yang L, Zheng Z, Li Y, Ren W, Wu C, Guo L. Calcitonin gene-related peptide induces IL-6 expression in RAW264.7 macrophages mediated by mmu_circRNA_007893. Mol Med Rep. 2017 Dec;16(6):9367-9374. doi: 10.3892/mmr.2017.7779. Epub 2017 Oct 12.</citation>
    <PMID>29039515</PMID>
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    <citation>Gallelli L, Siniscalchi A, Carotenuto M, Caroleo MC, Cione E, Guidetti V. microRNAs-based Predictor Factor in Patients with Migraine-ischemic Stroke. Microrna. 2017;6(1):17-21. doi: 10.2174/2211536606666170104130101.</citation>
    <PMID>28056747</PMID>
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    <citation>Ferroni P, Barbanti P, Spila A, Fratangeli F, Aurilia C, Fofi L, Egeo G, Guadagni F. Circulating Biomarkers in Migraine: New Opportunities for Precision Medicine. Curr Med Chem. 2019;26(34):6191-6206. doi: 10.2174/0929867325666180622122938.</citation>
    <PMID>29932027</PMID>
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    <citation>Headache Classification Committee of the International Headache Society (IHS) The International Classification of Headache Disorders, 3rd edition. Cephalalgia. 2018 Jan;38(1):1-211. doi: 10.1177/0333102417738202. No abstract available.</citation>
    <PMID>29368949</PMID>
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    <citation>Cernuda-Morollon E, Larrosa D, Ramon C, Vega J, Martinez-Camblor P, Pascual J. Interictal increase of CGRP levels in peripheral blood as a biomarker for chronic migraine. Neurology. 2013 Oct 1;81(14):1191-6. doi: 10.1212/WNL.0b013e3182a6cb72. Epub 2013 Aug 23.</citation>
    <PMID>23975872</PMID>
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    <citation>Andersen HH, Duroux M, Gazerani P. Serum MicroRNA Signatures in Migraineurs During Attacks and in Pain-Free Periods. Mol Neurobiol. 2016 Apr;53(3):1494-1500. doi: 10.1007/s12035-015-9106-5. Epub 2015 Feb 1.</citation>
    <PMID>25636687</PMID>
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    <PMID>29504482</PMID>
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    <PMID>25340934</PMID>
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    <citation>Lee MJ, Lee SY, Cho S, Kang ES, Chung CS. Feasibility of serum CGRP measurement as a biomarker of chronic migraine: a critical reappraisal. J Headache Pain. 2018 Jul 13;19(1):53. doi: 10.1186/s10194-018-0883-x.</citation>
    <PMID>30006780</PMID>
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    <citation>Munksgaard SB, Ertsey C, Frandsen E, Bendtsen L, Tekes K, Jensen RH. Circulating nociceptin and CGRP in medication-overuse headache. Acta Neurol Scand. 2019 Mar;139(3):269-275. doi: 10.1111/ane.13053. Epub 2018 Dec 11.</citation>
    <PMID>30457160</PMID>
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    <citation>Fan PC, Kuo PH, Lee MT, Chang SH, Chiou LC. Plasma Calcitonin Gene-Related Peptide: A Potential Biomarker for Diagnosis and Therapeutic Responses in Pediatric Migraine. Front Neurol. 2019 Jan 24;10:10. doi: 10.3389/fneur.2019.00010. eCollection 2019.</citation>
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    <citation>Frederiksen SD, Bekker-Nielsen Dunbar M, Snoer AH, Deen M, Edvinsson L. Serotonin and Neuropeptides in Blood From Episodic and Chronic Migraine and Cluster Headache Patients in Case-Control and Case-Crossover Settings: A Systematic Review and Meta-Analysis. Headache. 2020 Jun;60(6):1132-1164. doi: 10.1111/head.13802. Epub 2020 Apr 15.</citation>
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  <verification_date>July 2020</verification_date>
  <study_first_submitted>July 13, 2020</study_first_submitted>
  <study_first_submitted_qc>July 15, 2020</study_first_submitted_qc>
  <study_first_posted type="Actual">July 16, 2020</study_first_posted>
  <last_update_submitted>July 16, 2020</last_update_submitted>
  <last_update_submitted_qc>July 16, 2020</last_update_submitted_qc>
  <last_update_posted type="Actual">July 20, 2020</last_update_posted>
  <responsible_party>
    <responsible_party_type>Sponsor</responsible_party_type>
  </responsible_party>
  <keyword>Migraine</keyword>
  <keyword>CGRP</keyword>
  <keyword>MicroRNA</keyword>
  <keyword>Overuse of medications</keyword>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Migraine Disorders</mesh_term>
  </condition_browse>
  <patient_data>
    <sharing_ipd>No</sharing_ipd>
  </patient_data>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT04473976
org study id: CGRP-microRNA2020
nct id: NCT04473976
lead sponsor: 
collaborator: 
has dmc: No
is fda regulated drug: No
is fda regulated device: No
Migraine can manifest with an episodic or chronic pattern in a continuum of disease severity.
      Multiple factors are associated to the transformation of the pattern form episodic to
      chronic. Of these, the most consistently reported is the overuse of medications (MO) for the
      acute treatment of attacks. Knowledge of the mechanisms through which MO facilitates the
      transformation of episodic migraine (EM) into chronic migraine (CM) is very limited. In order
      insights into these mechanisms, the present study was aimed at identifying possible
      peripheral biomarkers associated to the 2 forms of migraine and to the presence of MO.

      The investigators evaluated CGRP plasma levels and the expression of miR-34a-5p and
      miR-382-5p in peripheral blood mononuclear cells of subjects with episodic migraine (EM,
      n=27) and CM-MO (n=28). CM-MO group was also tested 2 months after an in-hospital
      detoxification protocol.
Chronic migraine (CM) is a highly disabling condition that frequently manifests as a negative
      evolution of episodic migraine (EM) taking place over years. In a recent meta-analysis on the
      predictors of migraine chronification, a number of monthly headache days >10 showed the
      strongest level of evidence, with depression and low household income being supported by
      moderate evidence. In the same meta-analysis, medication overuse (MO) was associated to the
      highest risk ratio in a random-effects model (RR 8.82; 95% Confidence Interval (CI), 2.88-27)
      but the strength of evidence was rated 'very low'due to substantial heterogeneity among
      studies.

      It is likely that CM is the result of the dynamic interaction of multiple co-factors acting
      on a substrate represented by a more aggressive type of migraine. This hypothesis would
      explain why as many as 75% of CM subjects can spontaneously fluctuate around chronicity and
      episodicity. On the other hand, multiple pieces of evidence show that withdrawal from overuse
      medications induces a clinically meaningful improvement in a large percentage of subjects,
      with rates of benefit that are too high to be ascribed to a simple placebo effect.

      The mechanisms underlying the negative effect of MO in migraine outcome are largely unknown.
      Neural mechanisms including a combination of nociceptive facilitation with weakened
      descending pain inhibition, associated with trigeminal hyperexcitability, may be involved.
      The loss of diffuse descending inhibition has been demonstrated in a preclinical model of CM
      with MO. In this frame, it is worth noting that the investigators have recently reported a
      marked derangement of the endocannabinoid system in subjects with CM and MO (CM-MO), which
      was more marked when compared with subjects suffering from episodic migraine (EM).
      Interestingly, detoxification induced in CM-MO subjects the normalization of pain thresholds
      paralleled by the reduction in headache frequency. Pre-clinical studies show that triptans,
      the specific symptomatic migraine drugs, induce a condition of hyperalgesia when administered
      chronically. Thus, it is possible that acute migraine medications taken in repeated episodes
      of pain may amplify the consequences of nociceptor activation and increase the probability of
      subsequent migraine attacks, together with the risk of medication overuse.

      In this context of multiple concurrent causes, it seems extremely important to investigate in
      more depth the mechanisms that may be involved in CM and MO. Calcitonin gene-related peptide
      (CGRP) undoubtedly plays an important, though not exclusive, role in the generation of
      migraine headaches. CGRP receptors are localized in the sites involved in migraine
      pathogenesis. CGRP is involved in mast cell degranulation, neurogenic inflammation, and
      vasodilation. It has been shown that CGRP induces InterLeukin-6 (IL-6) gene expression in
      macrophages by upregulation of circular RNA_007893, a modulator of MicroRNA-485-5p. MicroRNAs
      (miRNAs) are involved in the generation and maintenance of chronic pain and several lines of
      evidence suggest that specific miRNAs may play a role in migraine pain. In a previous
      clinical study, Andersen and colleagues found an increased expression of miR-34a-5p during
      migraine attacks, while miR-382-5p levels increased also in the attack-free phase. In
      addition, the peripheral expression of miR-34a-5p decreased in the saliva of young
      migraineurs patients under drug treatment, thus suggesting a possible role in the prediction
      of therapeutic response. At present, no reliable individual biomarker of migraine and its
      subtypes has been identified, though multiple molecules have been proposed and supported by
      promising results.

      In this study, the investigators assayed the plasma levels of CGRP and the expression of
      miRNAs in PBMCs of patients with EM and CM-MO in order to identify individual or a panel of
      potential biomarkers of migraine subtypes. As secondary outcome, the investigators evaluated
      the changes in CGRP and miRNAs levels after detoxification in the subjects with CM-MO in
      order to gather more insights into the mechanisms that are involved in the improvement of
      migraine pattern following the withdrawal of the overused medications.

      This is a cross-sectional observational controlled study with 2 groups (EM and CM-MO),
      integrated with a prospective open label interventional trial to assess the effect of
      detoxification in the CM-MO group on the biomarkers of interest. Samples were labeled with
      numerical codes and all biochemical determinations were performed by researchers who were
      blind to the diagnosis.

      At baseline (Day 0), all patients underwent a visit with a neurologist of the Headache
      Science Centre during which clinical/demographical data were recorded and inclusion/exclusion
      criteria were checked. If the criteria were met, subjects underwent peripheral venipuncture
      for the evaluation of CGRP, miR-382-5p, and miR-34a-5p levels.

      Patients with CM-MO were hospitalized for a 7-day standardized detoxification protocol,
      consisting in abrupt withdrawal of overused drugs associated to intravenous therapy twice
      daily (08:00 a.m. and 4:00 p.m.) with isotonic 0.9% Sodium Chloride (NaCl) saline 500 ml +
      cyanocobalamin 2500 mcg + folic acid 0.70 mg + nicotinamide 12 mg + ascorbic acid 150 mg +
      sodic glutathione 600 mg + delorazepam 0.5 mg. Two months after hospital discharge (Day 1),
      the CM-MO patients returned for a follow-up visit, during which clinical data were recorded
      and a second venous blood sample obtained from their ante-cubital vein.
observational model: Other
time perspective: Cross-Sectional
measure: Plasma levels of CGRP
time frame: Day 0 at the enrolment
description: In order to identify individual or a panel of potential biomarkers of migraine subtypes
measure: Expression of miRNAs in PBMCs
time frame: Day 0 at the enrolment
description: In order to identify individual or a panel of potential biomarkers of migraine subtypes
measure: Changes in CGRP levels
time frame: Day1 - Two months after Day 0
description: In the subjects with CM-MO after detoxification in order to gather more insights into the mechanisms that are involved in the improvement of migraine pattern following the withdrawal of the overused medications.
measure: Changes in miRNAs levels
time frame: Day 1 - Two months after Day 0
description: In the subjects with CM-MO after detoxification in order to gather more insights into the mechanisms that are involved in the improvement of migraine pattern following the withdrawal of the overused medications.
Blood samples from the cubital vein of each patient was put into a 5 ml lavender tube. Plasma
      samples were prepared by centrifugation and then stored at -80°C. Plasma CGRP levels were
      measured using a commercial enzyme-linked immuno-sorbent assay kit.

      The miRNAs expression was evaluated by real-time reverse transcription (RT) Polymerase Chain
      Reaction (PCR) in peripheral blood mononuclear cell (PBMCs). PBMCs were isolated immediately
      after blood collection. Blood samples were collected within ethylenediamine tetra-acetic acid
      (EDTA) containing tube and diluted in 1:1 ratio with Phosphate Buffer Saline (PBS). Then,
      diluted blood samples were slowly loaded into Ficoll separating solution (15 ml) (Sigma) and
      centrifuged at 800 g for 30 min at room temperature. PBMCs accumulated as the middle white
      monolayer, were washed twice in sterile PBS at 300 g for 15 min. After washing, PBMCs pellet
      was resuspended in trizol and stored at -80 °C until use.
study pop: 
sampling method: Non-Probability Sample
criteria: 
gender: All
minimum age: 18 Years
maximum age: N/A
healthy volunteers: No
last name: Cristina Tassorelli, Prof
role: Principal Investigator
affiliation: IRCCS Mondino Foundation, Pavia
facility: 
country: Italy
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responsible party type: Sponsor
mesh term: Migraine Disorders
sharing ipd: No