PLOS ONE RESEARCH ARTICLE Exposure to valproic acid is associated with less pulmonary infiltrates and improvements in diverse clinical outcomes and laboratory parameters in patients hospitalized with COVID-19 Julio Collazos1, Pere Domingo2, Nerio Fernández-Araujo3, Elia Asensi-Dı́az4, a1111111111 Helem Vilchez-Rueda5, Antonio Lalueza ID6, Emilia Roy-Vallejo7, Rosa Blanes ID8, a1111111111 Manuel Raya-Cruz9, Jaime Sanz-Cánovas10, Arturo Artero ID11, José-Manuel Ramos- a1111111111 Rincón12, Carlos Dueñas-Gutiérrez13, José Luis Lamas-Ferreiro14, Vı́ctor Asensi ID15*, a1111111111 the Valproic Acid in COVID-19 Study Group¶ a1111111111 1 Infectious Diseases Section, Hospital de Galdakao-Usansolo, Galdakao, Vizcaya, Spain, 2 Infectious Diseases Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 3 Intensive Care Unit, Hospital General Universitario Gregorio Marañón, Madrid, Spain, 4 Internal Medicine Service, Hospital Universitario Fundación Jiménez Dı́az, Madrid, Spain, 5 Infectious Diseases Section, Hospital Universitari Son Espases, Palma de Mallorca, Spain, 6 Internal Medicine Service, Hospital 12 de Octubre, Madrid, Spain, OPEN ACCESS 7 Infectious Diseases Section, Hospital Universitario La Princesa, Madrid, Spain, 8 Internal Medicine Service, Hospital Universitario y Politécnico La Fe, Valencia, Spain, 9 Internal Medicine Service, Hospital Citation: Collazos J, Domingo P, Fernández-Araujo Son Llàtzer, Palma de Mallorca, Spain, 10 Internal Medicine Service, Hospital Regional Universitario, N, Asensi-Dı́az E, Vilchez-Rueda H, Lalueza A, et al. Málaga, Spain, 11 Internal Medicine Service, Hospital Universitario Dr Peset, Valencia, Spain, 12 Internal (2022) Exposure to valproic acid is associated with Medicine Service, Hospital General Universitario, Alicante, Spain, 13 Infectious Diseases Unit, Hospital less pulmonary infiltrates and improvements in Clı́nico Universitario, Valladolid, Spain, 14 Internal Medicine Service, Hospital POVISA, Pontevedra, Spain, diverse clinical outcomes and laboratory 15 Infectious Diseases Unit, Hospital Universitario Central de Asturias, Oviedo, Spain parameters in patients hospitalized with COVID-19. PLoS ONE 17(1): e0262777. https://doi.org/ ¶ Membership of the Valproic Acid in COVID-19 Study Group is listed in the Acknowledgments. 10.1371/journal.pone.0262777 * vasensia@gmail.com Editor: Mrinmoy Sanyal, Stanford University School of Medicine, UNITED STATES Received: July 24, 2021 Abstract Accepted: January 3, 2022 Published: January 27, 2022 Background Copyright: © 2022 Collazos et al. This is an open Valproic acid (VPA) has shown beneficial effects in vitro against SARS-CoV-2 infection, but access article distributed under the terms of the no study has analyzed its efficacy in the clinical setting. Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original Methods author and source are credited. This multicenter, retrospective study included 165 adult patients receiving VPA at the time Data Availability Statement: The data for this of admission to hospital, and 330 controls matched for sex, age and date of admission. A study were obtained from the 14 participating number of clinical, outcome and laboratory parameters were recorded to evaluate differ- hospitals, which belong to the Spanish National ences between the two groups. Four major clinical endpoints were considered: development Health System, a third-party organization. Data are available on request to interested researchers by of lung infiltrates, in-hospital respiratory worsening, ICU admissions and death. writing to: Dr Gonzalo Solis Presidente del Comite Ético de la Investigacion del Principado de Asturias Results Avenida de Roma s/n 33011 Oviedo,Spain Email: ceim.asturias@asturias.org. The authors had no VPA-treated patients had higher lymphocyte (P<0.0001) and monocyte (P = 0.0002) counts, special access privileges others would not have. and lower levels of diverse inflammatory parameters, including a composite biochemical PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 1 / 15 PLOS ONE Valproic acid improves COVID evolution Funding: The authors received no specific funding severity score (P = 0.016). VPA patients had shorter duration of symptoms (P<0.0001), for this work. were more commonly asymptomatic (P = 0.016), and developed less commonly lung infil- Competing interests: The authors have declared trates (65.8%/88.2%, P<0.0001), respiratory worsening (20.6%/30.6%, P = 0.019) and ICU that no competing interests exist. admissions (6.1%/13.0%, P = 0.018). There was no difference in survival (84.8%/88.8%, P = 0.2), although death was more commonly related to non-COVID-19 causes in the VPA group (36.0%/10.8%, P = 0.017). The cumulative hazard for developing adverse clinical endpoints was higher in controls than in the VPA group for infiltrates (P<0.0001), respiratory worsening (P<0.0001), and ICU admissions (P = 0.001), but not for death (0.6). Multivariate analysis revealed that VPA treatment was independently protective for the development of the first three clinical endpoints (P = 0.0002, P = 0.03, and P = 0.025, respectively), but not for death (P = 0.2). Conclusions VPA-treated patients seem to develop less serious COVID-19 than control patients, accord- ing to diverse clinical endpoints and laboratory markers. Introduction In December 2019 a cluster of severe pulmonary infections requiring hospital admission and, in many cases, mechanical ventilation in workers of the wet market of Wuhan, Hubei prov- ince, China, was reported [1]. These pulmonary infections were due to a new beta-coronavirus, named SARS-CoV-2, responsible for the so called COVID-19. Since then this infection became pandemic. As for November 7, 2021, 248 million of patients with confirmed COVID- 19 had been reported worldwide, including 5 million of deaths [2]. The COVID-19 pandemic has collapsed the health services of most of the countries throughout the world, even of the wealthiest, and ruined the world economy. In spite of intensive efforts by the pharmaceutical companies to obtain an effective antiviral drug against COVID-19, most of the drugs tested so far proved ineffective or even harmful such as hydroxychloroquine, azithromycin or lopinavir/ ritonavir [3–5]. Remdesivir, an antiviral previously designed to treat Ebola virus, is the only licensed drug with some antiviral effect on COVID-19. Remdesivir treatment seems to reduce the hospital admission days, but neither affects mortality nor decreases the viral load from nasopharyngeal or pulmonary exudates [6, 7]. So far, only dexamethasone has proved clinical benefit in reducing deaths in certain subgroups of patients [8] and fluvoxamine, a serotonin reuptake inhibitor, in reducing hospitalization among high-risk outpatients with early infec- tion [9]. Molnupiravir might become the first oral antiviral COVID treatment in the coming months. This antiviral, a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine (also called EIDD-1931), forces the SARS-CoV-2 to mutate itself to death by introducing copy- ing errors during viral RNA replication [10]. Valproic acid (VPA) is a branched, short-chain fatty acid, and the 2-n-propyl derivative of valeric acid. VPA has been used for more than 60 years to treat epilepsy and other neurologic and psychiatric diseases including bipolar disorders, neuropathic pain and migraine. In the last years an antiviral effect of VPA on herpes viruses, including herpes simplex virus 1 and 2, varicella zoster virus, Epstein-Barr virus and cytomegalovirus, has been reported [11–16]. Very recently an antiviral effect of VPA on dengue and SARS-CoV-2 has also been suggested in a virtual simulation study [17]. Interestingly, VPA administration decreased the LPs- PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 2 / 15 PLOS ONE Valproic acid improves COVID evolution induced lung inflammation in murine models, suggesting a protective effect of VPA on acute lung injury [18, 19], and suppressed TNF-alpha and IL-6 production via inhibition of NF-kap- paB activation, pointing towards a modulating role of VPA on immune responses [20]. VPA also inhibits histone deacetylase 2 (HDAC2), responsible for the deacetylation of lysine residues on the N-terminal region of the core histones, and HDAC2 inhibition modifies gene transcription. Thus VPA inhibits the expression of angiotensin-converting enzyme (ACE-2) receptors that are the entry door for SARS-CoV-2 into the cells [21]. Likewise, VPA decreases the expression of IL-6 in endothelial cells, a key element of the cytokines storm, the inflammatory event leading to lung infiltrates and pulmonary insufficiency in severe COVID- 19, and also decreases the expression of the intercellular adhesion molecule 1(ICAM-1), a cell surface glycoprotein that enhances viral adhesion to the capillary endothelial cells, and facili- tates intravascular thrombosis, a severe complication of COVID-19 [21]. A recent study using diverse cell lines found that VPA blocks three important processes involved in the severity of COVID-19 infection: the drug downregulates the expression of ACE2 and neuropili-1 (NRP1) decreasing viral infectivity, it reduces viral yields, probably because of alterations of the virus budding or virions stability, and it diminishes the inflamma- tory response to the viral infection [22]. Thus VPA is in an excellent position to be tested in patients with COVID-19 due to its anti- viral and inflammatory properties. However, no study to date has evaluated the possible use- fulness of VPA in the clinical setting. Because many individuals are exposed for years to VPA to treat their neuropsychiatric dis- orders, the easiest way to test VPA efficacy on COVID-19 is to analyze the presentation, evolu- tion and outcome of COVID-19 in patients who are taking VPA at the time of admission to the hospital. In this line of thought, we have carried out a retrospective, observational, multi- center study to compare the clinical and lab characteristics, course, treatments and outcomes of patients exposed and not exposed to VPA, matched by age, sex, and COVID-19 pandemic wave. Patients and methods This retrospective, case control study was developed in the following 14 Spanish hospitals, totalizing 10,917. hospital beds and providing care for a population of 4,612,712 inhabitants, about one-tenth of the total population of our country: Hospital de la Santa Creu i Sant Pau (Barcelona), Hospital General Universitario Gregorio Marañón (Madrid), Hospital Universi- tario Fundación Jiménez Dı́az (Madrid), Hospital Universitario Central de Asturias (Oviedo), Hospital Universitari Son Espases (Palma de Mallorca), Hospital Universitario 12 de Octubre (Madrid), Hospital Universitario La Princesa (Madrid), Hospital Universitario y Politécnico La Fe (Valencia), Hospital Son Llàtzer (Palma de Mallorca), Hospital Regional Universitario (Málaga), Hospital Clı́nico Universitario (Valladolid), Hospital General Universitario (Ali- cante), Hospital Universitario Dr Peset (Valencia), and Hospital POVISA (Vigo). Patients were considered for inclusion if admitted to the participating hospitals during a period of one year, from 1 March 2020 to 28 February 2021, covering the three major epidemic waves suffered in Spain from 2020 to 2021 (spring, autumn and winter). Cases were identified through electronic records. The first step was the detection of all in-hospital VPA prescriptions by means of the Pharmacy departments of the respective hospitals. The patients thus identified were scrutinized for simultaneous SARS-CoV-2 infection by means of microbiology records, PCR, antigen and serology results, codified diagnoses and/or discharge reports. Adult patients fulfilling the criteria of being treated with VPA at the time of arrival, SARS-- CoV-2 infection and hospital admission were included in the study as cases. Two controls PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 3 / 15 PLOS ONE Valproic acid improves COVID evolution were selected for each case matched by age, sex and date of admission, as each of these three features might have influence on the outcome. Particular attention was devoted to the choice of the controls to prevent selection bias. Thus, control patients were selected at each participat- ing hospital from electronic, non-clinical records, in which only the date of birth, gender and date of admission were available. Therefore, no clinical, laboratory or outcome data were known at the time of selection of the controls. The two closest patients to the index VPA case according to these three matching factors were selected as controls. A number of demographic, comorbid, microbiological, clinical, diagnostic, laboratory, imaging, hospitalization, prognostic, and therapeutic data were collected from medical records and compared in cases and controls. As many patients lacked diverse biochemical determina- tions, a composite biochemical severity score was constructed to evaluate the impact of the infection from a biochemical point of view in those parameters commonly affected by COVID-19 (C-reactive protein, alanine aminotransferase, lactic dehydrogenase, ferritin, pro- calcitonin, interleukin-6 (IL-6), D-dimer, troponin and N-terminal pro B-type natriuretic pep- tide (NT-proBNP)). This biochemical score was calculated for each patient by dividing the available determinations into their respective upper normal ranges, adding the resulting val- ues, and dividing this result into the number of parameters measured. Patients were also classified into two categories according to their respiratory evolution dur- ing the in-hospital stay. Worsening respiratory status was considered if the patients required additional oxygen supplementation or mechanical ventilation in the clinical ward or Intensive Care Unit (ICU) relative to their requirements at the time of arrival at the Emergency Depart- ment, whereas no worsening was considered if the need for oxygen supplementation did not increase during hospitalization. Four major clinical endpoints were considered for analysis: development of lung infiltrates, in-hospital respiratory worsening, ICU admission, and death. This was a retrospective, observational study using anonymized data from electronic rec- ords, based on patients who had undergone routine clinical care for COVID-19. Therefore, according to the Spanish law, no formal written informed consent was obtained from the patients, who had been already discharged from the hospital by then and some of whom even had died. The study was approved by the Research Ethics Committee of the Principality of Asturias, Spain, which also granted a formal waiver of requiring the consent from the patients. Statistical analysis As the distribution of continuous variables was non-Gaussian, according to the Kolmogorov- Smirnov test, original values underwent natural logarithmic transformation for analysis. The reported values are the result of back-transformation into the original units, and are expressed as geometric mean and 95% CI. Variables not suitable for logarithmic transformation, such as those containing 0 or negative values, are reported as median, IQ range. The t-test and the Mann-Whitney U test were used for the comparison of continuous variables, according to the nature of the variable. Proportions were compared with the chi-square test or Fisher’s exact test, as appropriate. Correlations among doses and duration of VPA treatment with other parame- ters were assessed with the Pearson’s correlation coefficient. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUROC) were calculated to identify the labo- ratory parameters more discriminant of clinical endpoints, whereas the differential association between cases and controls with these endpoints was analyzed with the Kaplan-Meier hazard function, using the log-rank Mantel-Cox test to assess their statistical significance. Logistic regression analysis models were constructed to identify the factors independently associated with clinical outcomes. SPSS v. 25 software (IBM Corp., Armonk, NY, USA) was used for statis- tical calculations. A P value <0.05 for a two-tailed test was considered statistically significant. PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 4 / 15 PLOS ONE Valproic acid improves COVID evolution Results A total of 165 cases and 330 controls were included in the study. The mean age was 60.1 years (95% CI 58.3–61.9) and the male to female ratio was 1.3/1. Regarding the epidemic waves, 159 patients (32.1%) were admitted during the first, 213 (43.0%) during the second and 123 (24.9%) during the third wave. We also calculated the incidences of hospital admissions by dividing the corresponding cases (165 for VPA patients and 43,637 for overall COVID-19 patients) into the total population covered by the 14 participating hospitals (4,612,712). Thus, the estimated incidence of hospital admissions because of COVID-19 of the VPA-treated patients during the year of study was 3.58/100,000 individuals, whereas the overall incidence of COVID-19 cases admitted to the participant hospitals during the same period was 946/ 100,000 individuals. Therefore, admissions of VPA-treated patients represented 0.38% of the total admissions for COVID-19. Table 1 shows the demographic features, habits, comorbidities and treatments at the hospi- tal arrival. There were no significant differences between the two groups in any of the parame- ters evaluated, with the only exception of a somewhat higher frequency of antihypertensive treatment with angiotensin receptor blockers in the control group, which was only marginally significant. However, this treatment was not significantly associated with the development of lung infiltrates (P = 0.3), respiratory worsening (P = 0.2), ICU admission (P = 0.2) or death (P = 0.9). Table 2 depicts the clinical features, radiological findings, laboratory determinations at admission and diagnostic procedures for SARS-CoV-2 infection. As compared to VPA cases, controls were more likely to be symptomatic and to have pulmonary infiltrates. Controls had also longer duration of symptoms and time to infiltrates detection and higher values of CRP, transaminases, ferritin and the biochemical severity score than the VPA group. Regarding hematological parameters, controls had significantly lower lymphocyte, monocyte and baso- phil counts than VPA-treated patients. The severity biochemical score was also significantly increased in patients with lung infil- trates (mean 3.63, 95% CI 3.35–3.94, vs 1.92 95% CI 1.56–2.36, P<0.0001), respiratory worsen- ing (mean 3.87, 95% CI 3.37–4.46 vs 2.96, 95% CI 2.68–3.25, P = 0.003), ICU admission (mean 5.49, 95% CI 4.66–6.47 vs 2.98, 95% CI 2.73–3.25, P<0.0001) and in those who died (mean 5.82, 95% CI 4.69–7.24 vs 2.93, 95% CI 2.69–3.18, P<0.0001). The AUROCs for these four out- comes were 0.687 (95% CI 0.622–0.752) P<0.0001; 0.588 (0.533–0.642) P = 0.003; 0.700 (0.638–0.761) P<0.0001; and 0.719 (0.646–0.793) P<0.0001, respectively. In multivariate anal- yses this biochemical severity score was independently predictive of the development of infil- trates (OR 1.641, 95% CI 1.161–2.319, P = 0.005), ICU admission (OR 1.939, 1.030–3.649, P = 0.04) and death (OR 2.361, 1.331–4.190, P = 0.003), but not of respiratory worsening (P = 0.2). VPA daily dosage only correlated with higher lymphocyte (n = 161, r = 0.20, P = 0.01) and mononuclear cell counts (n = 161, r = 0.19, P = 0.017), whereas duration of VPA treatment correlated with higher procalcitonin levels (n = 69, r = 0.30, P = 0.01), lower nasopharyngeal viral load (n = 7, r = -0.80, P = 0.029), and lower aspartate aminotransferase levels (n = 96, r = -0.24, P = 0.02). Dosage of VPA was not significantly associated with the development of lung infiltrates (P = 0.3), respiratory worsening (P = 0.9), ICU admission (P = 0.2) or death (P = 0.6). Table 3 shows the management and treatments during the hospital stay. There were no sig- nificant differences in the management of the two groups, with the exception of a significantly higher rate of dexamethasone treatment, as well as a trend towards a higher rate of tocilizumab therapy, in controls as compared to VPA patients. PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 5 / 15 PLOS ONE Valproic acid improves COVID evolution Table 1. Demography, habits, comorbidities and treatment at the time of hospital arrival. All VPA cases Controls P value (n = 495) (n = 165) (n = 330) Demography & anthropometry Gender Male 282 (57.0%) 94 (57.0%) 188 (57.0%) 1 Female 213 (43.0%) 71 (43.0%) 142 (43.0%) Age years (n = 495) 60.1 (58.3–61.9) 59.9 (56.5–62.8) 60.3 (58.2–62.6) 0.8 Weight kg (n = 279) 77.3 (75.4–79.2 77.2 (73.6–80.9) 77.3 (75.1–79.6) 0.9 Body mass index (n = 232) 28.34 (27.62–29.07) 27.96 (26.59–29.41) 28.52 (27.70–29.37) 0.5 Comorbidities / habits Diabetes Yes 95 (19.2%) 30 (18.2%) 65 (19.7.0%) 0.7 No 400 (80.8%) 135 (81.8%) 265 (80.3%) Hypertension Yes 231 (46.7%) 68 (41.2%) 163 (49.4%) 0.09 No 264 (53.3%) 97 (58.8%) 167 (50.6%) Immunosuppression Yes 49 (9.9%) 19 (11.5%) 30 (9.1%) 0.4 No 446 (90.1%) 146 (88.5%) 300 (90.9%) Immunosuppression, causes� Cancer 28 (57.1%) 8 (42.1%) 20 (66.7%) 0.15 HIV 6 (12.2%) 4 (21.1%) 2 (6.7%) Immunosuppressors 9 (18.4% 3 (15.8%) 6 (20.0%) Other conditions 6 (12.2%) 4 (21.1%) 2 (6.7%) Smoking Yes 32 (6.5%) 15 (9.1%) 17 (5.2%) 0.09 No 461 (93.5%) 149 (90.0%) 310 (94.8%) Alcohol (>50 gr/d for �5 years) Yes 20 (4.1%) 7 (4.3%) 13 (4.0%) 0.9 No 472 (95.9%) 156 (95.7%) 316 (96.0%) Antiepileptic/psychoactive therapy� VPA doses mg/day (n = 165) - 1098 (1001–1196) - - VPA therapy duration years (n = 142) - 6.77 (5.75–7.79) - - Reason for VPA therapy Epilepsy - 84 (50.9%) - - Mental disorder - 75 (45.5%) - Other 6 (3.6%) Concomitant use of other psychoactive drugs Yes - 68 (41.2%) - - No - 97 (58.8%) - Antihypertensive therapy� Angiotensin-converting enzyme inhibitors Yes 75 (33.8%) 16 (23.9%) 59 (38.1%) 0.04 No 147 (66.2%) 51 (76.1%) 96 (61.9%) Angiotensin receptor blockers Yes 84 (37.8%) 21 (31.3%) 63 (40.6%) 0.19 No 138 (62.2%) 46 (68.7%) 92 (59.4%) Other antihypertensive drugs Yes 140 (63.1%) 43 (64.2%) 97 (62.6%) 0.8 No 82 (36.9%) 24 (35.8%) 58 (37.4%) VPA denotes Valproic acid. Values are expressed as mean (95% CI) or %. � Only in patients who fulfilled the condition. https://doi.org/10.1371/journal.pone.0262777.t001 Table 4 describes the in-hospital course and outcomes. Controls experienced significantly higher rates of ICU admissions and respiratory worsening during the hospital stay than VPA- treated patients. On the contrary, no differences in the overall death rate were observed between cases and controls, although deaths specifically related to COVID-19 causes were sig- nificantly less common in the VPA than in the control groups in those patients who died. PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 6 / 15 PLOS ONE Valproic acid improves COVID evolution Table 2. Clinical, radiologic, laboratory and diagnostic procedures at admission. All VPA cases Controls P value (n = 495) (n = 165) (n = 330) Clinical features Duration of symptoms� days (n = 472) 5.0 (2.0–9.0) 3.0 (1.0–7.0) 7.0 (3.0–10.0) <0.0001 Asymptomatic Yes 23 (4.6%) 13 (7.9%) 10 (3.0%) 0.016 No 472 (95.4%) 152 (92.1%) 320 (97.0%) Temperature ˚C (n = 488) 37.03 (36.90–37.16) 37.03 (36.88–37.19) 37.03 (36.84–37.21) 1 Respiratory rate per minute (n = 391) 19.80 (19.308–20.31) 20.24 (19.32–21.22) 19.59 (18.99–20.31) 0.2 Heart rate per minute (n = 486) 87.65 (86.11–89.22) 86.74 (83.99–89.57) 88.10 (86.24–90.00) 0.4 Oxygen saturation % (n = 491) 93.5 (93.0–94.0) 93.2 (92.4–94.1) 93.6 (93.1–94.2) 0.4 pO2 mm Hg (n = 243) 70.2 (67.4–73.1) 69.2 (64.1–74.8) 70.6 (67.4–74.1) 0.6 Need for supplementary oxygen Yes 282 (57.0%) 106 (64.2%) 176 (53.3%) 0.02 No 213 (43.0%) 59 (35.8%) 154 (46.7%) Pulmonary infiltrates Yes 390 (80.7%) 106 (65.8%) 284 (88.2%) <0.0001 No 93 (19.3%) 55 (34.2%) 38 (11.8%) Bilateral pulmonary infiltrates� Yes 328 (84.1%) 87 (82.1%) 241 (84.9%) 0.5 No 62 (15.9%) 19 (17.9%) 43 (15.1%) Time to infiltrates detection since the onset of days (n = 384) 6.0 (3.0–9.0) 5.0 (2.0–8.0) 7.0 (4.0–10.0) 0.002 symptoms� § Laboratory blood determinations Total leukocyte count cells/μL (n = 485) 6411.3 (6163.1– 6439.2 (5995.1– 6397.6 (6100.7– 0.9 6669.6) 6916.2) 6700.9) Neutrophils cells/μL (n = 485) 4521.4 (4303.0– 4249.4 (3888.9– 4662.9 (4393.0– 0.08 4750.8) 4643.3) 4949.4) Lymphocytes cells/μL (n = 485) 1033.3 (985.2–1083.7) 1193.7 (1089.6– 961.8 (911.4–1014.8) <0.0001 1307.7) Monocytes cells/μL (n = 485) 459.8 (434.4–486.8) 535.8 (483.9–593.1) 426.2 (398.5–455.9) 0.0002 Eosinophils cells/μL (n = 485) 7.73 (6.58–9.09) 9.73 (7.19–13.18) 6.90 (5.70–8.34) 0.049 Basophils cells/μL (n = 485) 8.89 (7.83–10.08) 13.42 (10.79–16.68) 7.24 (6.23–8.41) <0.0001 C-reactive protein mg/L (n = 474) 46.26 (41.28–51.85) 35.93 (29.09–44.39) 52.56 (46.02–60.02) 0.002 Procalcitonin ng/mL (n = 255) 0.10 (0.09–0.11) 0.10 (0.08–0.13) 0.10 (0.09–0.12) 0.9 Aspartate aminotransferase U/L (n = 357) 32.66 (30.49–34.99) 29.43 (26.07–33.21) 34.39 (31.64–37.38) 0.036 Alanine aminotransferase U/L (n = 453) 26.21 (24.49–28.06) 21.63 (19.09–24.51) 28.82 (26.63–31.20) 0.0001 Creatine kinase U/L (n = 298) 102.6 (91.4–115.1) 115.0 (90.9–145.5) 96.5 (85.2–109.3) 0.15 Lactate dehydrogenase U/L (n = 398) 293.6 (282.6–304.6) 280.4 (261.0–301.2) 299.4 (286.6–312.8) 0.11 Ferritin ng/mL (n = 295) 460.4 (409.2–518.0) 352.5 (286.8–433.1) 524.7 (455.6–604.4) 0.002 IL-6 pg/mL (n = 210) 18.75 (15.16–23.18) 18.85 (12.72–27.94) 18.70 (14.50–24.11) 0.9 D-dimer ng/mL (n = 392) 585.3 (524.5–648.8) 593.4 (489.2–719.8) 578.6 (508.99–657.7) 0.8 Troponin ng/L (n = 190) 9.27 (8.05–10.67) 9.60 (7.35–12.53) 9.13 (7.72–10.79) 0.7 NT-proBNP pg/mL (n = 132) 262.0 (199.3–344.3 278.5 (179.6–431.9) 254.1 (178.7–361.3) 0.8 Biochemical severity score (n = 484) 3.19 (2.94–3.45) 2.77 (2.37–3.24) 3.42 (3.12–3.74) 0.016 Diagnostic procedures for SARS-CoV-2 infection Nasopharyngeal PCR Yes 451 (94.0%) 154 (93.9%) 297 (94.0%) 1 No 29 (6.0%) 10 (6.1%) 19 (6.0%) Nasopharyngeal viral load� log copies/1000 cells 6.437 (6.010–6.864) 6.541 (5.877–7.204) 6.380 (5.798–6.961) 0.7 (n = 45) Other samples PCR Yes 4 (2.2%) 0 (0.0%) 4 (3.4%) 0.14 No 174 (97.8%) 61 (100.0%) 113 (96.6%) (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 7 / 15 PLOS ONE Valproic acid improves COVID evolution Table 2. (Continued) All VPA cases Controls P value (n = 495) (n = 165) (n = 330) Nasopharyngeal antigen Yes 67 (63.8%) 19 (59.4%) 48 (65.8%) 0.5 No 38 (36.2%) 13 (40.6%) 25 (34.2%) Positive IgM serology Yes 37 (39.4%) 12 (30.8%) 25 (45.5%) 0.15 No 57 (60.6%) 27 (69.2%) 30 (54.5%) Positive IgG serology Yes 45 (37.5%) 17 (34.7%) 28 (39.4%) 0.6 No 75 (62.5%) 32 (65.3%) 43 (60.6%) Positive IgM and/or IgG Yes 51 (42.9%) 18 (36.7%) 33 (47.1%) 0.3 No 68 (57.1%) 31 (63.3%) 37 (52.9%) VPA denotes Valproic acid. Values are expressed as mean (95% CI), §median (IQ range) or %. � Only in patients who fulfilled the condition. https://doi.org/10.1371/journal.pone.0262777.t002 These favorable effects on certain clinical endpoints were not due to the higher rate of asymptomatic patients at admission in the VPA treated group, because significant differences were also observed when the asymptomatic patients were excluded from the analysis for lung infiltrates (P<0.0001), respiratory worsening (P = 0.026), and ICU admissions (P = 0.015). ROC curves revealed that lymphocyte and monocyte counts had similar predictive values and were the most discriminant hematological cell types for the four clinical endpoints, as well as for VPA treatment. The combination of these two mononuclear cells was moderately but significantly discriminatory for the development of lung infiltrates (AUROC 0.661, 95% CI 0.598–0.725, P<0.0001), respiratory worsening (AUROC 0.584, 0.528–0.640, P = 0.004), ICU admissions (AUROC 0.677, 0.602–0.753, P<0.0001), and death (AUROC 0.604, 0.522– 0.687, P = 0.009), as well as to identify the VPA group (AUROC 0.642, 0.589–0.696, P<0.0001). Taking into account the somewhat better predictive value of the combination of these two mononuclear cell types, hazard functions were calculated for the development of the four clini- cal outcomes evaluated in case and control patients (Fig 1). As seen in the Figure, control patients had significantly higher cumulative hazards for these adverse outcomes than VPA-treated individuals across the mononuclear cell range for the development of lung infiltrates, respiratory worsening and ICU admissions, but not for death. Regarding biochemical parameters, control patients also had higher cumulative hazards than VPA cases according to the biochemical severity index, although the association was somewhat less marked: lung infiltrates, P = 0.03; respiratory worsening, P = 0.06; ICU admissions, P = 0.027; death, P = 0.4. To identify the factors independently predictive of each of the four clinical endpoints evalu- ated, backward stepwise logistic regression analyses were carried out, including as explanatory variables those parameters that were significantly associated with the respective clinical end- point in the univariate analyses, although VPA treatment was entered into the analysis for death despite the lack of univariate association. In these models, which adequately fitted the data according to the Hosmer–Lemeshow goodness-of-fit statistic, VPA treatment was inde- pendently protective for the development of lung infiltrates (OR 0.309, 95% CI 0.168–0.568, P = 0.0002), in-hospital respiratory worsening (OR 0.508, 95% CI 0.273–0.945, P = 0.03), and ICU admissions (OR 0.251, 95% CI 0.075–0.843, P = 0.025), whereas it was not predictive of death (P = 0.2). PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 8 / 15 PLOS ONE Valproic acid improves COVID evolution Table 3. In-hospital management and treatment. All VPA cases Controls P value (n = 495) (n = 165) (n = 330) Supplementary oxygen in clinical ward/ICU Yes 352 (71.1%) 126 (76.4%) 226 (68.5%) 0.07 No 143 (28.9%) 39 (23.6%) 104 (31.5%) Treatment Dexamethasone Yes 192 (38.9%) 53 (32.3%) 139 (42.1%) 0.03 No 302 (61.1%) 111 (67.7%) 191 (57.9%) Dexamethasone duration� days (n = 192) 7.45 (6.78–8.18) 7.06 (5.70–8.75) 7.60 (6.85–8.42) 0.5 Methylprednisolone Yes 141 (28.5%) 40 (24.2%) 101 (30.7%) 0.13 No 353 (71.5%) 125 (75.8%) 228 (69.3%) Methylprednisolone duration� days (n = 139) 3.92 (3.44–4.47) 4.67 (3.59–6.08) 3.66 (3.15–4.26) 0.1 Tocilizumab Yes 47 (9.5%) 10 (6.1%) 37 (11.2%) 0.07 No 448 (90.5%) 155 (93.9%) 293 (88.8%) Tocilizumab duration� days (n = 46) 1.16 (1.04–1.30) 1.41 (0.87–2.29) 1.10 (1.01–1.20) 0.07 Remdesivir Yes 29 (5.9%) 8 (4.8%) 21 (6.4%) 0.5 No 466 (94.1%) 157 (95.2%) 309 (93.6%) Remdesivir duration� days (n = 28) 5.04 (4.56–5.58 4.46 (3.40–5.84) 5.30 (4.77–5.88) 0.12 Low-molecular weight heparin (�0,5 mg/Kg/d) Yes 420 (84.8%) 140 (84.8%) 280 (84.8%) 1 No 75 (15.2%) 25 (15.2%) 50 (15.2%) Heparin duration� days (n = 418) 9.52 (8.81–10.29) 9.80 (8.51–11.28) 9.39 (8.55–10.30) 0.6 Other commonly used drugs Hydroxychloroquine Yes 112 (22.6%) 39 (23.6%) 73 (22.1%) 0.7 No 383 (77.4%) 126 (76.4%) 257 (77.9%) Azithromycin Yes 103 (20.8%) 29 (17.6%) 74 (22.4%) 0.2 No 392 (79.2%) 136 (82.4%) 256 (77.6%) Lopinavir/ritonavir Yes 53 (10.7%) 13 (7.9%) 40 (12.1%) 0.15 No 442 (89.3%) 152 (92.1%) 290 (87.9%) Ceftriaxone Yes 100 (20.2%) 38 (23.0%) 62 (18.8%) 0.3 No 395 (79.8%) 127 (77.0%) 268 (81.2%) VPA denotes Valproic acid. Values are expressed as median (IQ range) or %. � Only in patients who fulfilled the condition. https://doi.org/10.1371/journal.pone.0262777.t003 Discussion To our knowledge, this is the first clinical study that evaluates the effect of VPA therapy on patients with COVID-19. We found that VPA-treated patients, as compared to control patients matched for age, sex and admission date, developed less commonly and had lower hazards for the development of pulmonary infiltrates, in-hospital respiratory worsening and ICU admissions, as well as had higher lymphocyte and monocyte counts and lower levels of diverse biochemical inflammatory markers. Beneficial effects that were confirmed by multivar- iate analyses after adjusting for covariates. Rates of ICU admissions should be interpreted cautiously, as diverse factors such as medical judgment, hospital policies and bed availability, among others, may influence this decision. However, our findings of a substantial reduction in ICU admissions in the VPA group seem consistent. Firstly, because these confounding factors would expectedly affect to both groups. Secondly, because of the degree of improvement, as control patients more than doubled the rate of ICU admission as compared to the VPA group. Finally, improvements in other clinical PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 9 / 15 PLOS ONE Valproic acid improves COVID evolution Table 4. In-hospital course and outcomes. All VPA cases Controls P value (n = 495) (n = 165) (n = 330) Nosocomial acquisition Yes 8 (1.6%) 6 (3.6%) 2 (0.6%) 0.01 No 487 (98.4%) 159 (96.4%) 328 (99.4%) Duration of in-hospital stay days (n = 495) 9.07 (8.41–9.77) 9.76 (8.49–11.21) 8.74 (8.00–9.55) 0.17 Time to discharge since the onset of symptoms� § days (n = 472) 16.0 (11.0–22.0) 15.0 (10.0–21.75) 16.0 (12.0–22.0) 0.07 Time to negative PCR since the onset of symptoms� § days (n = 159) 20.0 (14.0–32.0) 18.0 (13–29.5) 21.0 (14.0–33.0) 0.3 Respiratory worsening Yes 135 (27.3%) 34 (20.6%) 101 (30.6%) 0.019 No 360 (72.7%) 131 (79.4%) 229 (69.4%) Intensive Care Unit admission Yes 53 (10.7%) 10 (6.1%) 43 (13.0%) 0.018 No 442 (89.3%) 155 (93.9%) 287 (87.0%) Duration of ICU stay� days (n = 53) 14.06 (10.94–18.07) 17.45 (9.21–33.04) 13.35 (10.06–17.72) 0.4 Need for mechanical ventilation Yes 58 (11.7%) 14 (8.5%) 44 (13.3%) 0.11 No 437 (88.3%) 151 (91.5%) 286 (86.7%) Complications Yes 122 (24.6%) 43 (26.1%) 79 (23.9%) 0.6 No 373 (75.4%) 122 (73.9%) 251 (76.1%) Sequelae at discharge Yes 39 (8.9%) 16 (11.4%) 23 (7.7%) 0.2 No 399 (91.1%) 124 (88.6%) 275 (92.3%) Positive IgM serology at discharge Yes 66 (57.9%) 19 (47.5%) 47 (63.5%) 0.098 No 48 (42.1%) 21 (52.5%) 27 (36.5%) Positive IgG serology at discharge Yes 129 (75.4%) 46 (74.2%) 83 (76.1%) 0.8 No 42 (24.6%) 16 (25.8%) 26 (23.9%) Positive IgM and/or IgG at discharge Yes 129 (75.9%) 47 (75.8%) 82 (75.9%) 1 No 41 (24.1%) 15 (24.2%) 26 (24.1%) Outcome Survival 433 (87.5%) 140 (84.8%) 293 (88.8%) 0.2 Death 62 (12.5%) 25 (15.2%) 37 (11.2%) Death caused by COVID-19� Yes 49 (79.0%) 16 (64.0%) 33 (89.2%) 0.017 No 13 (21.0%) 9 (36.0%) 4 (10.8%) VPA denotes Valproic acid. Values are expressed as mean (95% CI), §median (IQ range) or %. � Only in patients who fulfilled the condition. https://doi.org/10.1371/journal.pone.0262777.t004 endpoints, such as development of lung infiltrates and respiratory worsening, as well as sub- stantial reductions in diverse hematological and biochemical parameters known to be associ- ated with severe COVID-19 were also objective indicators or a more benign disease in the VPA-treated patients. Despite these improvements in clinical endpoints and laboratory parameters, no improve- ment was observed in survival. However, it should be considered that all VPA-treated patients had additional neuropsychiatric comorbidities, some of them serious, which may account for additional deaths, both related and unrelated to COVID-19. In fact, we found that deaths not strictly related to-COVID-19 were significantly more common in the VPA than in the control group. In addition, control patients received more commonly than VPA patients some treat- ments aimed to improve COVID-19 outcomes, such as tocilizumab, methylprednisolone and, particularly, dexamethasone, a drug that has proved to be efficacious in reducing mortality rates [8], and that was administered significantly more frequently to control patients. Epilepsy was the most common cause of VPA treatment in our study, involving 51% of the patients. If patients with epilepsy suffer from more severe COVID-19 is controversial, as PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 10 / 15 PLOS ONE Valproic acid improves COVID evolution Fig 1. Hazard functions for the development of diverse clinical outcomes according to the blood mononuclear cell count (cells/μ). https://doi.org/10.1371/journal.pone.0262777.g001 exemplified by two large studies, one of which found that the infection was more prevalent, required more hospital and ICU admissions and was more lethal in epileptic patients than in the general population [23], whereas the other did not find differences between these two groups [24]. Low lymphocyte and monocyte counts have been associated with serious COVID-19 [25–30]. In this regard, we found significantly higher lymphocyte and monocyte and lower neutrophil counts in VPA-treated than in control patients, and there was also a positive cor- relation between VPA doses and lymphocyte counts, supporting the beneficial effects of such a therapy. A non-COVID-19 study also found higher lymphocyte and lower neutrophil counts in patients treated with VPA than in those treated with other antiepileptic drugs, although in this study there was a similar negative correlation between VPA plasma levels and both neutrophil and lymphocyte counts [31]. The preservation of the blood mononu- clear cells in VPA treated patients may have contributed to the clinical benefits observed in our study. Benefits of VPA treatment that are also supported by experimental studies [18, 19, 22] and by our findings of lower levels of some inflammatory biochemical parameters, including the biochemical severity score, as well as the lower hazards for developing certain PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 11 / 15 PLOS ONE Valproic acid improves COVID evolution adverse clinical endpoints according to the blood mononuclear counts. Therefore, the immunomodulatory and anti-inflammatory effects of VPA were presumably responsible for the improvements in these clinical endpoints. From our study we cannot derive which would be the optimal VPA dosage for obtaining the best results. Although there was a significant positive correlation between daily dosage and lymphocyte counts, indicating that higher doses were associated with higher improvements in lymphocyte counts, such association was only moderate, there is little correlation between the dose administered and serum levels due to its high plasma protein binding [32], and no signifi- cant relationship was found between dosage and clinical endpoints, suggesting that low VPA doses may be similarly efficacious as larger ones. Likewise, our study was based on VPA pre-treated patients and, therefore, the inferences about the effect that new treatments (e.g. at or before the onset of symptoms) could have on the course of the infection should be cautious. However, VPA is fully and rapidly absorbed, reaching peak plasma concentrations 1–4 hours after oral administration, and the steady state is reached 2–4 days after the onset of oral treatment, minutes in the case of intravenous administration [32, 33]. Therefore, its effects would expectedly be rapid, as also indicated by in vitro studies [18–22], and, consequently, its early administration might not only have a beneficial effect in hospitalized patients, but also a potential role in the prevention of hospi- tal admissions. Dexamethasone is nowadays the most effective therapy for serious COVID-19 due to its well-known anti-inflammatory properties. Treatment with this drug resulted in lower 28-day mortality in patients receiving either invasive mechanical ventilation or oxygen alone, but not in those who did not receive respiratory support [8]. On the other hand, the effect of the anti- IL-6 monoclonal antibody tocilizumab on patients hospitalized with COVID-19 is more con- troversial, although tocilizumab use may be associated with a short-term mortality and a reduction in the need for mechanical ventilation [34, 35]. Interestingly VPA-treated patients needed less dexamethasone and tocilizumab therapy, as well as methylprednisolone and remdesivir, compared to controls in our study, and had lower rates, not only of pulmonary infiltrates or respiratory worsening, but also ICU admission, an aspect that none of these drugs could demonstrate. The main limitations of this study are those related to retrospective studies, including the lack of certain laboratory data in many patients and the inferences regarding causality, as well as the limited number of VPA-treated patients and the small number of deaths, which pre- cluded us to perform a more precise mortality analysis. Also, we cannot entirely dismiss the existence of any occult VPA-unrelated factor that could favor the outcomes of the VPA-treated group, although this possibility is very unlikely and, in fact, these patients had additional comorbidities such as epilepsy, dementia and mental disorders. The main strengths of the study are its novelty and the enrollment of hospitalized patients throughout our country, cov- ering about one-tenth of the Spanish population and the three COVID-19 major waves that ours and other Western European countries have suffered so far. Conclusions We conclude that exposure to VPA seems to protect against the development of severe COVID-19, reducing the development of lung infiltrates, respiratory worsening and ICU admissions. This treatment was also associated with lower serum levels of diverse biochemical inflammatory markers, and higher peripheral blood lymphocyte and monocyte counts, objec- tive laboratory parameters that support the clinical observations. However, large, prospective studies are needed to further clarify the role of VPA on COVID-19. PLOS ONE | https://doi.org/10.1371/journal.pone.0262777 January 27, 2022 12 / 15 PLOS ONE Valproic acid improves COVID evolution Acknowledgments This work was done by the Valproic Acid in COVID-19 Study Group whose leading member is Dr Julio Collazos, email: med0053033@gmail.com Other members of the Valproic Acid in COVID-19 Study Group are: Virginia Pomar (Hospital de la Santa Creu i Sant Pau, Barcelona); Silvia Manrique-Rodrı́- guez and Irene Taladriz-Sender (Hospital General Universitario Gregorio Marañón, Madrid); Raquel Bravo-Ruiz, Paula Asensio-Matthews, Barbara Soler-Bonafont, and Marina Bernal- Palacios (Fundación Jiménez Dı́az, Madrid); Sara Rodrı́guez-Suárez, Laura Pérez-Is, Carlos López-Larrea, Miguel Alaguero, and Alberto Garcı́a (Hospital Universitario Central de Astu- rias, Oviedo); Adrián Ferre-Beltrán (Hospital Universitari Son Espases, Palma de Mallorca); Elisa Cerezo-Benichou (Hospital 12 de Octubre, Madrid); Aresio Sancha-Lloret and Jesús Sanz-Sanz (Hospital Universitario La Princesa, Madrid); Marino Blanes (Hospital Universi- tario y Politécnico La Fe, Valencia); Lidia Cobos-Palacios, Almudena López-Sampalo, Rosa M. Bernal-López and Ricardo Gómez-Huelgas (Hospital Regional Universitario, Málaga); Ana Martı́n-Pastor and Rosario Sánchez-Martı́nez (Hospital General Universitario, Alicante); Sara Gutiérrez-González, Laura Rodrı́guez-Fernández and Genoveva Zapico-Aldea (Hospital Clı́n- ico Universitario, Valladolid); Javier de la Fuente (Hospital POVISA, Pontevedra). Author Contributions Conceptualization: Julio Collazos, Vı́ctor Asensi. Data curation: Julio Collazos, Pere Domingo, Nerio Fernández-Araujo, Elia Asensi-Dı́az, Helem Vilchez-Rueda, Antonio Lalueza, Emilia Roy-Vallejo, Rosa Blanes, Manuel Raya- Cruz, Jaime Sanz-Cánovas, Arturo Artero, José-Manuel Ramos-Rincón, Carlos Dueñas- Gutiérrez, José Luis Lamas-Ferreiro, Vı́ctor Asensi. Formal analysis: Julio Collazos. Investigation: Julio Collazos, Vı́ctor Asensi. Methodology: Julio Collazos, Vı́ctor Asensi. Supervision: Julio Collazos, Vı́ctor Asensi. Validation: Julio Collazos, Vı́ctor Asensi. 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