Background: Calcitonin gene-related peptide (CGRP) is the most promising candidate to become the first migraine biomarker. However, literature shows clashing results and suggests a methodological source for such discrepancies. We aimed to investigate some of these methodological factors to evaluate the actual role of CGRP as biomarker. Methods: Previous to the experimental part, we performed a literature review of articles measuring CGRP in migraine patients. Using our 399 bio-bank sera samples, we performed a series of experiments to test the validity of different ELISA kits employed, time of sample processing, long-term storage, sampling in rest or after moderate exercise. Analysis of in-house data was performed to analyse average levels of the peptide and the effect of sex and age. Results: Literature review shows the high variability in terms of study design, determination methods, results and conclusions obtained by studies including CGRP determinations in migraine patients. CGRP measurements depends on the method and specific kit employed, also on the isoform detected, showing completely different ranges of concentrations. Alpha-CGRP and beta-CGRP had median with IQR levels of 37.5 (28.2–54.4) and 4.6 (2.4–6.4)pg/mL, respectively. CGRP content is preserved in serum within the 24 first hours when samples are stored at 4°C after clotting and immediate centrifugation. Storages at -80°C of more than 6 months result in a decrease in CGRP levels. Moderate exercise prior to blood extraction does not modulate the concentration of the peptide. Age positively correlates with beta-CGRP content and men have higher alpha-CGRP levels than women. Conclusions: We present valuable information for CGRP measurements in serum. ELISA kit suitability should be tested prior to the experiments. Alpha and beta-CGRP levels should be analysed separately as they can show different behaviours even within the same condition. Samples can be processed in a 24-h window if they have been kept in 4°C and should not be stored for more than 6 months at -80°C before assayed. Patients do not need to rest before the blood extraction unless they have performed a high-endurance exercise. For comparative studies, sex and age should be accounted for as these parameters can impact CGRP concentrations. Graphical

Objective: To analyze the specificity of calcitonin gene–related peptide (CGRP) levels, we measured alpha‐CGRP circulating levels in a large series of patients with a recent diagnosis of inflammatory bowel disease (IBD) who were interviewed regarding comorbid headache. Background: Several studies have found an association between migraine and IBD. Methods: In this cross‐sectional study performed in an IBD clinic, morning serum alpha‐CGRP levels were measured by enzyme‐linked immunosorbent assay in 96 patients who were recently diagnosed with IBD and compared to those from 50 similar patients with chronic migraine (CM) and 50 healthy controls (HC). Results: Alpha‐CGRP levels were higher in patients with IBD (median [interquartile range] 56.9 [35.6–73.9] pg/mL) and patients with CM (53.0 [36.7–73.9] pg/mL) compared to HC (37.2 [30.0–51.8] pg/mL; p = 0.003; p = 0.019, respectively). Regarding IBD diagnostic subtypes, alpha‐CGRP levels for ulcerative colitis (67.2 ± 49.3 pg/mL; 57.0 [35.6–73.4] pg/mL) and Crohn's disease (54.9 ± 27.5 pg/mL; 57.7 [29.1–76.1] pg/mL) were significantly higher than those of HC ( p = 0.013, p = 0.040, respectively). Alpha‐CGRP levels were further different in patients with IBD with migraine (70.9 [51.8–88.7] pg/mL) compared to HC ( p < 0.001), patients with IBD without headache (57.5 [33.3–73.8] pg/mL; p = 0.049), and patients with IBD with tension‐type headache but without migraine (41.7 [28.5–66.9] pg/mL; p = 0.004), though alpha‐CGRP levels in patients with IBD without migraine (53.7 [32.9–73.5] pg/mL) remained different over HC ( p = 0.028). Conclusion: Together with CM, circulating alpha‐CGRP levels are different in patients with IBD, perhaps reflecting a chronic inflammatory state. IBD is an example of how alpha‐CGRP levels are not a totally specific migraine biomarker. However, alpha‐CGRP levels were further increased in patients with IBD who have a history of migraine, which reinforces its role as a biomarker in migraine patients, always bearing in mind their comorbidities.

Background: Fremanezumab was the third CGRP antibody available in our hospital. This examination of our experience with fremanezumab is focused on identifying the predictors of response. Methods: This was a prospective observational study in which we included high-frequency episodic/chronic migraine (HF/CM) patients who were prescribed fremanezumab during the year 2023. Our research involved collecting data on their demographic details, diagnoses made, treatments received, prophylactic measures taken in the past, and any comorbid conditions present. The number of headaches was documented for one quarter prior to and after the initiation of fremanezumab. Results: Eighty-nine patients received fremanezumab (86.5% female, 45.8 ± 12.5 years old, 70.1% naive). The headache days decreased from 21.1 ± 7.6 to 12.4 ± 11.2 days during the initial three months of the treatment, and a total of 55 patients (61.8%) exhibited a response rate of ≥50%. Six out of ten patients refractory to erenumab for at least 6 months responded to fremanezumab. Totals of 17 and 26 patients had been treated at least with galcanezumab or erenumab. The elements influencing non-response were as follows: prior failure to respond to both erenumab and galcanezumab (p < 0.0001), HF/CM length (11.9 ± 7.1 years in non-responders vs. 5.8 ± 4.8 in responders; p < 0.001), the presence of fibromyalgia (p < 0.001), anxiety–depression (p < 0.001), an almost daily headache baseline (>28 days/month) (p < 0.0001), and analgesic overuse (p < 0.0001). The response rate was unaffected by age and experience. After a multivariate logistic analysis, almost daily headaches (p < 0.001), a length of HF/CM > 6 years (p = 0.015), and anxiety–depression (p = 0.017) remained significant. Fremanezumab showed excellent tolerance. Conclusions: These real-life results confirm the efficacy of fremanezumab. The main factors associated with a lack of response were almost daily/daily headaches and a disease duration > 6 years. Half of the patients who failed to respond to erenumab improved on fremanezumab, making it sensible to switch to a treatment with a different mechanism of action, but trying a third anti-CGRP treatment in patients with no response to both a receptor-targeted and a ligand-targeted CGRP antibody hardly seems justifiable from our experience.

Background: Serum 25‐hydroxyvitamin D (25[OH]D) concentrations have been shown to be low in patients with migraine, but results are controversial regarding the current role of vitamin D in migraine severity. Using a case–control design, we aimed to evaluate serum 25(OH)D levels in a group of females with high‐frequency episodic migraine/chronic migraine (HF/CM) and analyze its association with headache frequency and serum calcitonin gene–related peptide (CGRP) levels.Methods: Serum 25(OH)D levels were measured in 97 females with HF/CM (age 48.9 ± 9.4 years) and 146 healthy females (47.4 ± 8.1 years). Participants taking vitamin D supplements were excluded. Serum concentrations of 25(OH)D were determined by electrochemiluminescence (Roche, Germany), and CGRP levels were measured by enzyme‐linked immunosorbent assay (Abbexa, UK).Results: Mean 25(OH)D levels in females with HF/CM (median [interquartile range] 19.0 [13.0–24.5] ng/mL) were below the values considered for insufficiency or deficiency and significantly lower than controls (25.0 [19–29.8] ng/mL; p < 0.0001). Fifty (51.5%) patients with HF/CM had levels below 20 ng/mL. There was no significant association between vitamin D levels and monthly headache days (Spearman's rank correlation coefficient [rho]: −0.086; p = 0.404) or with serum α (rho: 0.114; p = 0.267) and β‐CGRP (rho: 0.113; p = 0.276) levels. Serum 25(OH)D levels in females with HF/CM with a minimum daily sunlight exposure were significantly higher than those without (23.0 [15.0–26.0] ng/mL vs. 14.0 [10.0–20.0] ng/mL; p < 0.001). Females with HF/CM who performed exercise had higher, albeit not significant, plasma 25(OH)D levels than those who did not (21.0 [15.5–28.0] ng/mL vs. 16.5 [12.0–24.0] ng/mL; p = 0.059).Conclusions: Serum concentrations of 25(OH)D were low in many patients with HF/CM. Because there was no correlation with migraine frequency or serum CGRP levels, this deficiency seems to be a direct consequence of the migraine impact. Our data do not support either a relationship of 25(OH)D levels with migraine severity or the use of vitamin D supplements as a specific migraine treatment, although further studies are needed.