Supplementary Materials for

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1 Supplementary Materials for A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch Christopher E. Ramsden,* Anthony F. Domenichiello, Zhi-Xin Yuan, Matthew R. Sapio, Gregory S. Keyes, Santosh K. Mishra, Jacklyn R. Gross, Sharon Majchrzak-Hong, Daisy Zamora, Mark S. Horowitz, John M. Davis, Alexander V. Sorokin, Amit Dey, Danielle M. LaPaglia, Joshua J. Wheeler, Michael R. Vasko, Nehal N. Mehta, Andrew J. Mannes, Michael J. Iadarola This PDF file includes: *Corresponding author. chris.ramsden@nih.gov Published August 07, Sci. Signal. 0, eaal54 (07) DI: 0.6/scisignal.aal54 Fig. S. Proton NMR data for authentic standards. Fig. S. Identification of hydroxy-epoxy-octadecenoate and keto-epoxyoctadecenoates. Fig. S3. Basal and low ph evoked CGRP release in response to lipid mediators. Table S. Rat tissue concentrations of hydroxy-epoxy- and keto-epoxyoctadecenoates. Table S. Concentrations of hydroxy-epoxy- and keto-epoxy-octadecenoates in control skin and psoriatic skin. Table S3. Percentage of total hydroxy-epoxy- and keto-epoxy-octadecenoates as free acids in control skin and psoriatic skin. Table S4. Serum concentrations of free hydroxy-epoxy- and keto-epoxyoctadecenoates in control and psoriatic patients. Table S5. Association between diet-induced changes in plasma linoleic acid derivatives and pain-related end points in the CDH trial. Table S6. Mass spectrometry parameters established for the smrm mode. Table S7. Demographic and clinical characteristics of psoriatic patients and controls. Table S8. Baseline demographic and clinical characteristics of patients with CDH.

2 Figure S. Proton NMR data for authentic standards. For H 400 MHz NMR spectra, tetramethylsilane or the solvent peak served as an internal standard for reporting chemical shifts, expressed on the δ scale. H NMR spectra acquired on a Varian Inova 400MHz NMR. Standards prepared by total organic synthesis. -hydroxy-,3-trans-epoxy-(9z)-octadecenoic acid (H-,3E-LA): H NMR (400 MHz, CDCl3) 5.59 (td, J=7.48,.0 Hz, H), (m, H), and 4.5 (tdd, J=.37, 5.5, 8.58 Hz, H), (m, H), 3.0 (dt, J=.0, 5.49 Hz, H), (m, H), (m, H),.3 (dt, J=.37, 7.46 Hz, H),.98-. (m, H),.-.68 (m, 8H), (m, 3H) -hydroxy-9,0-trans-epoxy-(z)-octadecenoic acid (H-9,0E-LA): H NMR (400 MHz, CDCl3) 5.63 (tt, J=7.46,.39 Hz, H), (m, H), 4.66 (dd, J=.84, 8.69 Hz) and 4.8 (ddd, J=0.9, 5.35, 8.74 Hz, H), 3.0 (dt, J=.38, 5.58 Hz, H),.9 (dt, J=.38, 5.67 Hz, H), (m, H),.34 (t, J=7.4 Hz, H), (m, H), (m, 5H),.-.46 (m, 6H), 0.88 (t, J=6.77 Hz, 3H)

3 9-hydroxy-,3-trans-epoxy-(0E)-octadecenoic acid (9H-,3E-LA): H NMR (400 MHz, CDCl3) 5.9 (dd, J=6., 5.55 Hz, H), 5.4 (dd, J=7.87, 5.55 Hz, H), 4. (quin, J=5.95 Hz, H), (m, H),.8 (dt, J=.9, 5.54 Hz, H),.33 (t, J=7.50 Hz, H), (m, 5H), (m, H), (m, 3H), (m, 3H) 3-hydroxy-9,0-trans-epoxy-(E)-methyl-octadecenoate (3H-9,0E-LA methyl ester): H NMR (400 MHz, CDCl3) 5.9 (dd, J=6.40, 5.55 Hz, H), 5.40 (dd, J=7.87, 5.55 Hz, H), (m, H), 3.65 (s, 3H), 3.08 (dd, J=.0, 7.78 Hz, H),.80 (dt, J=.0, 5.58 Hz, H),.9 (t, J=7.50 Hz, H),.69 (br. s., H), (m,6h), (m, 3H), 0.87 (t, J=6.59 Hz, 3H) -keto-,3-trans-epoxy-(9z)-methyl-octadecenoate (K-,3E-LA methyl ester): H NMR (400 MHz, CDCl3) (m, H), (m, H), 3.65 (s, 3H), 3.9 (d, J=.83 Hz, H), 3.0 (dt, J=.0, 5.49 Hz, H), (m, H),.8 (t, J=7.5 Hz, H), (m, 9H), (m, H), (m, 3H)

4 -keto-9,0-trans-epoxy-(z)-methyl-octadecenoate (K-9,0E-LA methyl ester): H NMR (400 MHz, CDCl3) 6.6 (td, J=7.30,.57 Hz, H), (m, H), 3.66 (s, 3H), 3.0 (d, J=.65 Hz, H), (m, H),.64 (q, J=7.44 Hz, H), (m, H), (m, 5H),.-.49 (m, 6H), 0.87 (br t, J=6.86 Hz, 3H) 9-keto-,3-trans-epoxy-(0E)-methyl-octadecenoate (9K-,3E-LA methyl ester): H NMR (400 MHz, CDCl3) (m, H), (m, H), 3.7 (s, H), (m, 3H), 3.0 (d, J=6.77 Hz, H), (m, H), (m, 3H),.-.33 (m, 3H), (m, 0H),.-.38 (m, 0H), (m, 3H) 3-keto-9,0-trans-epoxy-(E)-octadecenoic acid (3K-9,0E-LA): H NMR (400 MHz, CDCl3) (m, H), (m, H), 3.0 (dd, J=.83, 6.86 Hz, H),.89 (dt, J=.0, 5.58 Hz, H), (m, H), (m, H), (m, 4H), (m, H),.-.38 (m, H), (m, 3H)

5 Figure S. Identification of hydroxy-epoxy-octadecenoate and keto-epoxy-octadecenoates. A total of eleven metabolites of four hydroxy-epoxy-octadecenoates and four keto-epoxyoctadecenoates were found in human psoriatic skin samples. All these metabolites exhibited an abundant [M-H]ˉ at 3 and 309 for hydroxy-epoxy-octadecenoates and keto-epoxyoctadecenoates, respectively in ESI negative mode. The MS/MS spectra of hydroxy-epoxyoctadecenoates and keto-epoxy-octadecenoates are presented in Figs. SA-H, left panel, along with the detailed fragmentation analysis of these compounds. Figs. SA-H, right panel show the extracted MRM chromatograms from synthetic/authentic standards and human psoriatic skin samples. Five out of seven synthesized standards showed two or three isomeric peaks that had almost identical MS/MS spectra. The metabolites M to M were identified through comparison of MS/MS spectra of metabolites with those of synthetic or authentic standards and further confirmed by matching retention times to the corresponding standard via MRM analysis. Metabolites M and M eluted at retention time 4.6 and 5.4 min from human psoriatic skin samples have been identified with fragment ions at m/z 7, 39, 7, 85, 0, 75, and 93 as H-9,0E-LA isomers (Fig. SA). M3 and M4 eluted at 4.8 and 5.5 min and have been identified with fragment ions at m/z 9, 69, 8, 97,, 75, and 93 as H-,3E-LA isomers (Fig. SB). M and M4 coeluted; therefore mixed MS/MS spectra were observed that contain fragment ions from both. M5 eluted at 4.3 min has been identified with fragment ions at m/z 39, 53 7,, 75, and 93 as 3H-9,0E-LA and M6 eluted at 4.6 min and has been identified with fragment ions at m/z 39, 7, 93,, 75, and 93 as 9H-,3E-LA, respectively (Figs. SC and D). Two isomeric metabolites M7 and M8 eluted at 4.8 and 5.5 have been identified with fragment ions at m/z 55, 67, 7, 85, 47, and 9 as K-9,0E-LA corresponding to its isomers observed at 9.9 and. min and the third one eluted at 3.0 was not found (Fig. SE). nly one isomeric peak M9 eluted at 9.5 min with fragment ions at m/z 5, 65, 8, 9, 09, 47, 65, 9 has been identified as K-,3E-LA (Figure S6). M0 eluted at 9.3 min with fragment ions at m/z 37, 55, 7, and 93, 65, and 9 has been identified as 3-K-9,0E-LA (Fig. SG), although two and three isomeric peaks in synthesized standard were observed for K-,3E-LA and 3K-9,0E-LA, respectively (Fig. SF and G). M eluted at 8.5 min showed fragment ions at m/z 5, 39, 65, 93, 09, 47, and 9 has been identified as 9K-,3E-LA (Fig. SH).

6 -H -H 3 4 M M (A) Identification of metabolite M and M as (+/-)--hydroxy-(z)-9,0-trans-epoxyoctadecenoate (H-9,0E-LA) isomers. Left Panel: MS/MS spectra of () Synthetic standard H-9,0E-LA at 4.6 min, () Synthetic standard H-9,0E-LA at 5.4 min, (3) M, and (4) M; Right Panel: LC-MS/MS MRM chromatograms for transition 3>0 () Synthetic standard H-9,0E-LA; and () a human psoriatic skin sample.

7 -H -H 3 4 M3 M4 (B) Identification of metabolites M3 and M4 as (+/-)--hydroxy-(9z)-,3-trans-epoxyoctadecenoate (H-,3E-LA) isomers. Left Panel: MS/MS spectra of () Synthetic standard H-,3E-LA at 4.8 min, () Synthetic standard H-,3E-LA at 5.5 min, (3) M3, and (4) M4; Right Panel: LC-MS/MS MRM chromatograms for transition 3>97 () Synthetic standard H-,3E-LA; and () a human psoriatic skin sample.

8 -H -H M5 (C) Identification of metabolite M5 as (+/-)-3-hydroxy-(E)-9,0-trans-epoxy-octadecenoate (3H-9,0E-LA). Left Panel: MS/MS spectra of () Synthetic standard 3H-9,0E-LA at 4.3 min, () M5; Right Panel: LC-MS/MS MRM chromatograms for transition 3>7 () Synthetic standard 3H-9,0E-LA; and () a human psoriatic skin sample.

9 -H -H M6 (D) Identification of metabolite M6 as (+/-)-9-hydroxy-(0E)-,3-trans-epoxy-octadecenoate (9H-,3E-LA). Left Panel: MS/MS spectra of () Synthetic standard 9H-,3E-LA at 4.6 min, () M6; Right Panel: LC-MS/MS MRM chromatograms for transition 3>93 () Synthetic standard 9H-,3E-LA; and () a human psoriatic skin sample.

10 -H -C -H -HCH 3 4 M7 5 3 M8 (E) Identification of metabolite M7 and M8 as (+/-)--keto-9,0-trans-epoxy-(z)- octadecenoate (K-9,0E-LA) isomers. Left Panel: MS/MS spectra of () Synthetic standard K-9,0E-LA at 9.9 min, () Synthetic standard K-9,0E-LA at. min, (3) Synthetic standard K-9,0E-LA at 3.0 min, (4) M7 (5) M8; Right Panel: LC-MS/MS MRM chromatograms for transition 309>85 () Synthetic standard K-9,0E-LA; and () and (3) a human psoriatic skin sample.

11 -C -H -H 3 -C 4 M9 (F) Identification of metabolite M9 as (+/-)--keto-,3-trans-epoxy-(9z)-octadecenoate (K-,3E-LA) isomers. Left Panel: MS/MS spectra of () Synthetic standard K-,3E- LA at 9.5 min, () Synthetic standard K-,3E-LA at.7 min, (3) Synthetic standard K-,3E-LA at.5 min, (4) M9; Right Panel: LC-MS/MS MRM chromatograms for transition 309>8 () Synthetic standard K-,3E-LA; and () a human psoriatic skin sample.

12 -H -C -H -C 3 M0 (G) Tentative* identification of metabolite M0 as (+/-)-3-keto-9,0-trans-epoxy-(E)- octadecenoate (3K-9,0E-LA). Left Panel: MS/MS spectra of () Synthetic standard 3K- 9,0E-LA at 9.3 min, () Synthetic standard 3K-9,0E-LA at 0.3 min, (3) M0; Right Panel: LC-MS/MS MRM chromatograms for transition 309>7 () Synthetic standard 3K-9,0E-LA; and () a human psoriatic skin sample. *indicates that this identification is tentative and requires confirmation in additional studies.

13 -H -C -H -C M (H) Identification of metabolite M as (+/-)-9-keto-,3-trans-epoxy-(0E)-octadecenoate (9K-,3E-LA). Left Panel: MS/MS spectra of () Synthetic standard 9K-,3E-LA at 8.6 min, () M; Right Panel: LC-MS/MS MRM chromatograms for transition 309>65 () Synthetic standard 9K-,3E-LA; and () a human psoriatic skin sample.

14 Figure S3. Basal and low ph evoked CGRP release in response to lipid mediators. Ex vivo CGRP release was measured from adult rat DRG neuronal cultures in response to PGE or LAderived lipid mediators ( µm). Basal release was measured before addition of solution to the culture meda (blue), followed by addition of µm lipid mediator alone (green), then µm lipid mediator in the presence of ph 6.0 (red). Solutions were washed out and measured a final time (blue with hatching). N=9 wells (A) or wells (B), each from three separate harvests. Significance was determined using a one-way ANVA with Tukey s post-hoc test (*, p<0.05). Error bars represent the standard error of the mean. H-,3E-LA, -hydroxy-,3-epoxyoctadecenoate; H-9,0E-LA, -hydroxy-9,0-trans-epoxy-octadecenoate; K-,3E-LA, -keto-,3-trans-epoxy-octadecenoate; K-9,0E-LA, -keto-9,0-trans-epoxyoctadecenoate; 3H-9,0E-LA, 3-hydroxy-9,0-trans-epoxy-octadecenoate; 3K-9,0E-LA, 3-keto-9,0-trans-epoxy-octadecenoate. Normalized values for low ph + mediator data are also shown in Figure 3.

15 Table S. Rat tissue concentrations of hydroxy-epoxy- and keto-epoxy-octadecenoates. Hindpaw Median (IQR) Total (n=4) Median (IQR) Free (n=4) -hydroxy-,3-epoxy-octadecenoate.3 (8.7, 07.3) BLQ -hydroxy-9,0-epoxy-octadecenoate 53. (450.5, 660.8) BLQ 9-hydroxy-,3-epoxy-octadecenoate 45.0 (384.3, 648.5) 0.3 (9., 5.3) 3-hydroxy-9,0-epoxy-octadecenoate (3389.0, ) 0.9 (9.4, 9.5) -keto-,3-epoxy-octadecenoate BLQ BLQ -keto-9,0-epoxy-octadecenoate BLQ BLQ 9-keto-,3-epoxy-octadecenoate 83.0 (040., 977.) BLQ 3-keto-9,0-epoxy-octadecenoate 77.7 (85., 9.5) BLQ Dorsal Horn Total (n=7) Free (n=4) -hydroxy-,3-epoxy-octadecenoate BLQ BLQ -hydroxy-9,0-epoxy-octadecenoate BLQ BLQ 9-hydroxy-,3-epoxy-octadecenoate BLQ BLQ 3-hydroxy-9,0-epoxy-octadecenoate BLQ BLQ -keto-,3-epoxy-octadecenoate BLQ BLQ -keto-9,0-epoxy-octadecenoate BLQ BLQ 9-keto-,3-epoxy-octadecenoate BLQ BLQ 3-keto-9,0-epoxy-octadecenoate BLQ BLQ N=4 tissue specimens from independent harvests. IQR, interquartile range; BLQ, below the limit of quantitation. ng/g, nanograms per gram

16 Table S. Concentrations of hydroxy-epoxy- and keto-epoxy-octadecenoates in control skin and psoriatic skin. Free Control (n=7) Psoriasis Non Lesion (n=8) Psoriasis Lesion (n=8) Wilcoxon Median IQR Median IQR Median IQR Rank-sum* H-,3E-LA 7.48 (3.43, 45.5) (38.0, 69.37) (57.9, ) H-9,0E-LA.33 (0.67, 4.50) 3.58 (5.34, 95.4) (6.4, 93.43) H-,3E-LA BLQ 0.67 (7.34, 86.44) 4.37 (7.0, 54.47) H-9,0E-LA.58 (0.43,.8).37 (., 0.76).50 (5.30, 48.44) 0.0 K-,3E-LA BLQ BLQ BLQ K-9,0E-LA 8.33 (.5, 35.79) (4.80, 44.4) (4.59, ) K-,3E-LA 5.58 (3.7, 6.9) 30.4 (9., 3.83) 7.3 (68.54, ) K-9,0E-LA BLQ BLQ BLQ Total (free plus esterified) H-,3E-LA 6.4 (8.03, 97.95) 80.7 (38.08, 58.50) (45.0, 37.04) 0.48 H-9,0E-LA 4.3 (30.8, 5.8) (37.89, 49.58) 94.6 (43.9, 9.04) H-,3E-LA BLQ BLQ (48.84, 389.9) H-9,0E-LA (095.00, ) (8.03, ) (538.48, ) 0.98 K-,3E-LA (88.00, 94.9) (66.0, 84.9) (66.5, ) K-9,0E-LA ( , 735.9) 9 ( , ) (433.90, ) K-,3E-LA (775.00, 086.7).07 (808.55, 879.7) (77.65, 959.6) K-9,0E-LA (596.55, 9.60) (493.86, 379.5) 5.6 (57.37, 07.05) 0.03 Control skin samples are from 7 participants without psoriasis; psoriasis non-lesion and lesion samples are from different sites on 8 psoriasis patients. *indicates comparison of psoriasis lesion and control groups; BLQ values imputed as 50% of the limit of quantitation. ng/g, nanograms per gram. BLQ, below the limit of quantitation; H-,3E-LA, -hydroxy-,3-epoxyoctadecenoate; H-9,0E-LA, -hydroxy-9,0-epoxy-octadecenoate; 9H-,3E-LA, 9- hydroxy-,3-epoxy-octadecenoate; 3H-9,0E-LA, 3-hydroxy-9,0-epoxy-octadecenoate; K-,3E-LA, -keto-,3-epoxy-octadecenoate; K-9,0E-LA, -keto-9,0-epoxyoctadecenoate; 9K-,3E-LA, 9-keto-,3-epoxy-octadecenoate; 3K-9,0E-LA, 3-keto- 9,0-epoxy-octadecenoate

17 Table S3. Percentage of total hydroxy-epoxy- and keto-epoxy-octadecenoates as free acids in control skin and psoriatic skin. Control (n=7) Psoriasis Non Lesion (n=8) Psoriasis Lesion (n=8) Wilcoxon Median IQR Median IQR Median IQR H-,3E-LA 44.4 (3.80, 60.83) 07.7 (5.57, 80.6) (0.39, 37.00) 0.00 H-9,0E-LA 8.4 (.33, 47.88) 5.8 (5.95, 6.77) 55.9 (.8, 38.56) 0.3 3H-9,0E-LA 0.05 (0.04, 0.3) 0.9 (0.06, 0.9) 0.45 (0.8,.04) K-9,0E-LA 0.37 (0.6, 0.47) 0.35 (0.9, 5.0) 4.08 (0.77, 8.8) 0.0 Ranksum* 9K-,3E-LA 0.7 (0.8, 0.84).66 (0.77, 3.79) 3.7 (9.40, 0.) 0.00 Control skin samples were collected from 7 participants without psoriasis; psoriasis non-lesion and lesion samples were collected from different sites on 8 psoriasis patients. *Comparison of psoriasis lesion and control groups. IQR = interquartile range. H-,3E-LA, -hydroxy-,3-epoxy-octadecenoate; H-9,0E-LA, -hydroxy-9,0-epoxy-octadecenoate; 3H-9,0E- LA, 3-hydroxy-9,0-epoxy-octadecenoate; K-9,0E-LA, -keto-9,0-epoxy-octadecenoate; 9K-,3E-LA, 9-keto-,3-epoxy-octadecenoate

18 Table S4. Serum concentrations of free hydroxy-epoxy- and keto-epoxy-octadecenoates in control and psoriatic patients. Control (n=7) Psoriasis (n=8) Mediator (ng/ml) Median IQR Median IQR p-value* -hydroxy-,3-epoxy-octadecenoate 0.05 (0.04, 0.07) 0.06 (0.03, 0.) hydroxy-9,0-epoxy-octadecenoate 0.57 (0.47, 0.67) 0.5 (0.4, 0.56) hydroxy-,3-epoxy-octadecenoate 0.7 (0.4, 0.3) 0. (0., 0.3) hydroxy-9,0-epoxy-octadecenoate 0.39 (0.6, 0.5) 0.4 (0.37, 0.49) keto-9,0-epoxy-octadecenoate 0. (0., 0.3) 0. (0., 0.) keto-,3-epoxy-octadecenoate 0.70 (0.67, 0.75) 0.6 (0.59, 0.75) 0.45 *Wilcoxon rank-sum tests. ng/ml, nanograms per milliliter of serum

19 Table S5. Association between diet-induced changes in plasma linoleic acid derivatives and pain-related end points in the CDH trial. Compound PAIN FREQUENCY & INTENSITY FUNCTINAL DIMENSINS F PAIN PSYCHLGICAL DIMENSINS F PAIN Headache Headache HIT-6 3 SF- SF- BSI-8 5 hours/day days/month physical 4 mental 4 n=40 n=44 n=44 n=44 n=44 n=44 Coef p-value Coef p-value Coef p-value Coef p-value Coef p-value Coef p-value H-,3E-LA 5% (<0.00) % (<0.00) 0.8 (0.033) -0.3 (0.058) (0.8) -0.0 (0.96) H-9,0E-LA.% (0.867) 3.4% (0.384) -0.0 (0.90) (0.488) 0.07 (0.595) -0.4 (0.45) 3H-9,0E-LA -4.5% (0.537) -5% (<0.00) (0.508) 0 (0.99) 0. (0.359) (0.669) 9H-,3E-LA % (0.69) -4.% (0.383) (0.73) (0.595) 0.8 (0.59) -0.6 (0.66) 9K-,3E-LA.8% (0.68) 4.8% (0.99) 0.03 (0.80) -0. (0.36) 0. (0.359) -0.3 (0.73) Analyzed using Poisson regression and presented as % change in count for each standard deviation change in the respective fatty acid. Analyzed using linear regressions and presented as standard deviation change in Y for each standard deviation change in X. 3 Headache Impact Test. Higher score = more headache impact on quality of life. 4 Short Form (SF)- Health Survey. Higher score = better quality of life. 5 Brief Symptom Inventory (BSI)-8. Higher score = more psychological distress. Coef, coefficient; H-,3E-LA, -hydroxy-,3-epoxy-octadecenoate; H-9,0E-LA, -hydroxy- 9,0-epoxy-octadecenoate; 3H-9,0E-LA, 3-hydroxy-9,0-epoxy-octadecenoate; 9H-,3E-LA, 9- hydroxy-,3-epoxy-octadecenoate; 9K-,3-LA, 9-keto-,3-epoxy-octadecenoate

20 Table S6. Mass spectrometry parameters established for the smrm mode. Analytes Retention time (min) Q Q3 DP CE CXP IS H-9,0ELA LTB4-d4 3H-9,0E-LA LTB4-d4 9H-,3E-LA LTB4-d4 H-,3E-LA LTB4-d4 K-9,0E-LA HDE-d4 3K-9,0E-LA HDE-d4 9K-,3E-LA HDE-d4 K-,3E-LA HDE-d4 3-HDE-d LTB4-d DP, declustering potential; CE, collision energy; CXP, collision cell exit potential; IS, internal standard; H-9,0E-LA, -hydroxy-9,0-epoxy-octadecenoate; 3H-9,0E-LA, 3-hydroxy- 9,0-epoxy-octadecenoate; 9H-,3E-LA, 9-hydroxy-,3-epoxy-octadecenoate; H-,3E- LA, -hydroxy-,3-epoxy-octadecenoate; K-9,0E-LA, -keto-9,0-epoxyoctadecenoate; 3K-9,0E-LA, 3-keto-9,0-epoxy-octadecenoate; 9K-,3E-LA, 9-keto-,3-epoxy-octadecenoate;K-,3E-LA, -keto-,3-epoxy-octadecenoate

21 Table S7. Demographic and clinical characteristics of psoriatic patients and controls. Characteristics Psoriasis (N=8) Controls (N=7) p value Demographics and Medical History Age, years 57±6 43± Male, % 6 (75%) 6 (86%) 0.6 Ethnicity (whites), % 8 (00%) 7 (00%).00 Current smoking, % 0 (0%) 0 (0%).00 Alcohol use, % 0 (0%) 3 (43%) 0. Hypertension, % (5%) (3%) 0.88 Type II Diabetes mellitus, % (3%) (4%) 0.9 Hyperlipidemia, % 5 (63%) 4 (57%) 0.83 Body Mass Index, kg/m 3.4± ± Psoriasis related variables PASI score, (Median [IQR]) 6.5 ( ) BSA 3 score, (Median [IQR]) 4. (7.7-4.) Itch 4 (%) 5 (63%) Disease Duration (years) 3.75±6.63 Treatment Topical (%) 5 5 (63%) Systemic/Biologic (%) 0 (0%) t-test for continuous variables, Pearson s Chi-square for categorical variables. Values reported in the table as Mean ± SD or median (IQR) for continuous variables and as N (%) for categorical variables. indicates more 7 or more drinks per week for women and 4 or more drinks for men. Psoriasis Area and Severity Index (PASI) 3 Body Surface Area (BSA) affected by psoriatic lesions 4 substantial itch complaint in self-reported questionnaire 5 No topical medication use in the past weeks

22 Table S8. Baseline demographic and clinical characteristics of patients with CDH. H3-L6 intervention n=33 L6 intervention n=34 Age, years: Mean (SD) 4 (3.4) 4 (.) Female, n (%) 8 (84.8) 30 (88.) Married, n (%) 9 (57.6) 9 (55.9) Headache category, n (%) Chronic migraine 6 (78.8) 4 (70.6) CDH with migraine features 6 (8.) 6 (7.6) CDH without migraine features (3.0) 4 (.8) Headache Days per month, mean (95%CI) 3.3 (0.9, 5.8) 3. (0., 5.8) Headache Hours per day, mean (95%CI) 0. (8.4,.3) 9.8 (8.,.8) Headache Impact Test, mean (95%CI) 6.0 (59.5, 6.5) 60.6 (58.7, 6.6) Age at first headache, median (IQR) 5 (0) 8 (8) Total headache-related medications reported, mean (SD) 6.4 (3.4) 5.6 (3.3) Credibility Scale (Borkovec & Nau) (range 0 45), mean (SD) 8.3 (5.5) 7. (3.8) CDH, chronic daily headache; IQR, interquartile range; CI, confidence interval; SD, standard deviation. Subjects classified as chronic migraine met International Headache Disorders- criteria. Subjects classified as CDH with migraine features had some characteristics of migraine (unilateral, pulsating, severe, sensory sensitivity, or aggravated by physical activity) but did not meet all criteria needed for chronic migraine diagnosis. Subjects classified with CDH without migraine features had no evidence of migraine.