Ecacy and Safety of Pidotimod in Childhood Wheezing: A Pilot Study

Background. Effective therapies for the management of wheezing in pediatric population are needed. We conducted a pilot, mono-centre, prospective, follow-up study to assess the ecacy and the safety of Pidotimod (PDT) in the treatment of wheezing in children. Methods. Globally, 90 children (M:F=58:62, mean age 4.7±1.64 years) with recurrent viral wheezing were enrolled in the study between October-November 2018. At baseline, children were receiving treatment with PDT as 1 vial of 400mg daily for 3 consecutive months. We evaluated the therapeutic ecacy of PDT treatment at the end of 3 (T3) months of therapy as well as the long ecacy and preventive ecacy of PDT treatment during a 3-months follow-up (T6) by using the following outcomes: (i) how many patients showed one or more episodes of viral wheezing, (ii) how many patients were taking concomitant medications (ICS, SABA, antibiotics), (iii) how many patients were requiring ED visits, and (iv) how many patients were requiring hospitalization. Results. A signicant decrease in number of patients with at least one or more episodes of wheezing and taking antibiotics was recorded after 3 months of treatment, and a further signicant decrease for both outcomes was reported at 3-months follow-up period (p<0.05). Differently, after 3 months of treatment, we found a signicant decrease in number of patients taking ICS and SABA, in number of patients requiring ED visits and/or hospitalization (p<0.05); however, for all these outcomes no further signicant decrease was reported at follow-up period. Conclusions. We rstly showed that administration of PDT is useful in the management of patients with recurrent viral wheezing because we found a reducing a number of patients requiring ED visits and/or hospitalization as well as number of patients taking drugs during the treatment period. Moreover, rstly to date, we found a long-term clinical effect over three months after treatment suspension counteracting the recurrence of disease.

PDT as 1 vial of 400mg daily for 3 consecutive months. We evaluated the therapeutic e cacy of PDT treatment at the end of 3 (T3) months of therapy as well as the long e cacy and preventive e cacy of PDT treatment during a 3-months follow-up (T6) by using the following outcomes: (i) how many patients showed one or more episodes of viral wheezing, (ii) how many patients were taking concomitant medications (ICS, SABA, antibiotics), (iii) how many patients were requiring ED visits, and (iv) how many patients were requiring hospitalization.
Results. A signi cant decrease in number of patients with at least one or more episodes of wheezing and taking antibiotics was recorded after 3 months of treatment, and a further signi cant decrease for both outcomes was reported at 3-months follow-up period (p<0.05). Differently, after 3 months of treatment, we found a signi cant decrease in number of patients taking ICS and SABA, in number of patients requiring ED visits and/or hospitalization (p<0.05); however, for all these outcomes no further signi cant decrease was reported at follow-up period.
Conclusions. We rstly showed that administration of PDT is useful in the management of patients with recurrent viral wheezing because we found a reducing a number of patients requiring ED visits and/or hospitalization as well as number of patients taking drugs during the treatment period. Moreover, rstly to date, we found a long-term clinical effect over three months after treatment suspension counteracting the recurrence of disease.

Background
Wheezing is a common disorder in early life, affecting approximately 50% of children in the rst years of life. It represents a signi cant cause of morbidity, decreased quality of life, and even increased health care services and economic costs, due to the frequent emergency department (ED) visits and hospitalizations [1]. Genetic factors and the environment are well recognized causative factors for wheeze although they may vary from child to child and within a child over time [2]. Whether in accordance of its temporal pattern, wheezing is classi ed into transient versus persistent wheeze, in accordance with the causative agents, wheezing is classi ed into multiple-trigger and episodic or viral wheezing. The latter appears to be most common in preschool children and usually associated with clinical evidence of a viral respiratory tract infection generally sustained by rhinovirus, respiratory syncytial virus (RSV), coronavirus, human metapneumovirus, parain uenza virus, and adenovirus [3]. The management for acute episodes of virus-induced wheezing in infants and preschool children was established but the current therapeutic strategy on the clinical bene t is not evident. Due to the lack of a speci c treatment, researchers on viral wheezing in childhood have increased in recent years not reaching, however, conclusive data. Pidotimod (PDT), 3-L-pyroglutamyl-L-thiaziolidine-4-carboxylic acid), a synthetic thymic dipeptide, in uences both the innate and adaptive immune response. By causing maturation of mucosal dendritic cells, increasing antigen presentation, and promoting adaptive T helper (Th)1-mediated immunity, PDT induces the release of antimicrobial peptides and improves the mucociliary transport showing positive effects on the immune pro le of children suffering recurrent respiratory tract infections (RTIs) [4][5][6]. In light of these immunological properties, PDT was approved in Italy as bene cial immunomodulator agent in treating RTIs. Following its marketing, several studies assessed both the good e cacy, expressed in terms of reduced number of RTIs, shortening the symptom duration, and decreasing signs and symptoms severity, as well as safety of PDT in treatment of recurrent RTIs [7].
Taking in account the aetiology of wheezing and the evidence of the effects of PDT as an adjuvant in the treatment of recurrent viral RTIs, we hypothesized that PDT could prove to be a new therapeutic option in children suffering for recurrent viral wheezing. To achieve this goal, we conducted a pilot, mono-centre, prospective study with PDT in the treatment of viral wheezing in childhood.

Study design
A mono-centre, prospective, follow-up study was designed. Exclusion criteria included: children younger than 3 years, associate diseases (hepatic, infectious, or endocrine diseases, genetic syndromes, immunode ciencies, neurological and psychiatric problems), pulmonary or cardiac congenital malformations, chronic pulmonary disease (broncho-pulmonary dysplasia, cystic brosis, bronchiolitis obliterans post-infection), and congenital and/or acquired craniofacial anomalies. Patients receiving treatment with other immunomodulantors were also excluded.
The investigator collected the symptoms recorded by the parent in the study diary during the treatment period.
Institutional Review Board of the University of Catania approved the study. Written informed consent was obtained from the parents and informed assent from the children and adolescents.

Study procedure and e cacy evaluation
The patients were receiving PDT as one vial of 400 mg daily. Treatment lasted for 90 consecutive days and followed by 3-months follow-up period.
All enrolled patients were compared at baseline (T0) and three months (treatment period) (T3), and after the subsequent 3-months follow-up (T6). We evaluated how many patients showed one or more episodes of viral wheezing, how many patients were taking concomitant medications (inhaled corticosteroid (ICS); short-acting beta-agonists (SABA), antibiotics), how many patients were requiring ED visits, and how many patients were requiring hospitalization during the period of therapy. After the subsequent 3-months follow-up (T 6), we evaluated how many patients showed one or more episodes of viral wheezing, how many patients were taking concomitant medications (ICS, SABA, antibiotics), how many patients were requiring ED visits, and how many patients were requiring hospitalization.

Safety
Safety of PDT was evaluated as the number and the type (mild, moderate, and severe) of adverse events (AEs) recorded by a physician and/or parents children.

Data analysis
The data collected were statistically analyzed by the statistical computer software SPSS, version 15.0.
Values were calculated as mean and standard deviation, T-student and χ 2 tests were adopted for comparisons between variables. A p-value less than 0.05 were considered statistically signi cant.

Results
All 90 children (M:F = 47:43, mean age 4.7 ± 1.64 years) completed the study and were considered in the nal analysis. Clinical outcomes at T0, T3, and T6 were reported in Table 1.

Emergency department visits
After three months of treatment, a signi cant decrease in the number of patients requiring ED visits was recorded (p < 0.001; Table 1); no further signi cant decrease was reported at follow-up period (p = 1.00; Table 1).

Hospitalization
After 3 months of treatment, a signi cant decrease in the number of patients requiring hospitalization was recorded (p < 0.05; Table 1); no further signi cant decrease was reported at follow-up period (p = 1.00; Table 1).

Safety
PDT treatment was well tolerated and no signi cant adverse effects were reported.

Discussion
By conducting a pilot, mono-centre, prospective follow-up study, we rstly reported the therapeutic e cacy and safety of PDT in treating of childhood viral wheezing.
It is well known that wheezing is characterized by signi cant morbidity and represents a very common health problem in the rst years of life, resulting in a high burden to the affected patients and families who have a greater need for primary care, specialist physician visits, ED visits, and hospitalization [1].
Currently, maintenance treatment with ICS and/or montelukast are recommended for episodic or viral and multiple-trigger wheeze, respectively, as well as SABA by metered-dose inhaler/spacer combination, are suggested for symptomatic relief [1]. However, although the daily use of ICS and/or montelukast signi cantly reduces wheezing exacerbations, they do not prevent virus-induced wheezing attacks, and, to date, shadows remain on their use [8,9].
The shift of the immune balance to pro-/Th2/anti-Th1 direction, the increase both in the in ammatory response and Th2-related marker as well as in serum immunoglobulin (Ig)E levels and eosinophils in ltration are some of the mechanisms resulting in airway remodelling, wheezing, and asthma [1,10,11]. By acting on all these pathways [12][13][14][15], it has been hypothesized that PDT could prove to be a possible therapeutic approach in treating patients with wheezing. To the best of our knowledge, studies on the e cacy of PDT in patients affected by wheezing are relatively sparse and, the only studies identi ed did not report clear and exhaustive data since affected by methodological limitations including heterogeneity in enrolled population and some analysis, lack of su cient details on the clinical outcomes, and absence of follow-up period [16,17]. Firstly, in their papers, authors did not focus their investigations on wheezing as children with asthma and the related obstructive syndrome were enrolled; moreover, no data on number of patients with episodes of wheezing over time were described, thus, an objective outcome measure of PDT e cacy has not been reported [16,17]. Secondly, both studies compared the data of children of such different ages (2-16 years) [16,17]. In our pilot study, we restricted our investigations to children with the diagnosis of viral wheezing and restricted our investigation to children aged 3-6 years; further, all outcome measures of treatment e cacy were clearly stated.
Speci cally, data on the number of patients with at least one or more episodes of wheezing, number of patients taking concomitant drugs (ICS, SABA, antibiotics), number of patients requiring ED visits and hospitalizations were collected and all these outcomes appeared to be signi cantly decreased by PDT administration.
ICS therapy has been widely adopted in treating wheezing with bene t. In our study, 3-months treatment with PDT was associated with a signi cant decrease in the number of patients taking ICS, an outcome measure of therapy e cacy. ICS use in children has lagged because of concerns about the potential for adverse effects related to the dose, the frequency, and duration treatment with which ICS are administered. High dosage (≥ 1.000 ug/day), frequent administration (≥ 4 times /day) are more likely to have local, such as oral candidiasis and dysphonia, as well as systemic adverse effects [1]. The potential for growth suppression and osteoporosis with long-term ICS use has been also a concern, especially in children who receive maintenance therapy with higher ICS doses because they are also likely to require systemic CS use. In this regard, PDT treatment reduced the number of patients taking routine ICS also preventing indirectly the side effects associated with a frequent ICS. The improvement of disease control has been indirectly assessed also by a signi cant decrease in the number of patients taking SABA after three months of treatment with PDT. Inhaled salbutamol is recommended in emergency management in children with wheezing, thus, a decrease in the number of patients taking SABA re ects the e cacy of PDT treatment in preventing wheezing exacerbations. In parallel, a signi cant decrease both in the number of patients requiring ED visits and in the number of patients requiring hospitalizations was recorded after three months of PDT treatment, further con rming an improvement in health status due to the PDT administration. Lastly, although we did not perfom any estimate of the wheezing-related economic burden, we indirectly speculated on the evidence that PDT use, by decreasing signi cantly the use of concomitant drugs (ICS, SABA, antibiotics), ED visits and hospitalizations, can also contribute to reduce the economic burden of wheezing in the pediatric population.
Moreover, at 3-months follow-up period, a signi cant decrease in the number of patients taking antibiotics was also recorded. As viral agents are the causes of the episodic wheezing, they do not require antibiotic treatment. Nevertheless, antibiotic prescription occurs frequently. We explained the reduction in antibiotics use as an indirect sign of disease improvement; the patients and their parents perceived as less severe the episodes of wheezing, and, probably, a prescription of antibiotic was not required.
Follow-up completeness is a pre-requisite for reliable outcome assessment, and the lack of a follow-up period in the previous studies correlates inversely with the accuracy of outcome estimates, with the risk of selection bias, and with the credibility of study data [16,17]. Conversely, by adopting a follow-up period study, we also rstly provided data on long-term effects of PDT and observed signi cant changes in the course of the disease as assessed by a further decrease of the number of patients with at least one or more episodes of wheezing also at 3-months PDT suspension. Although 60% of children with wheezing are expected to improve and/or be symptom-free at the age of 6 years [2], children with wheezing are at higher risk of developing asthma at school age, thus, thanks to PDT use, we also believe that is possible to interfer with long-term outcomes of disease, even if it will be not possible to prevent evolution of wheezing in asthma. Further studies with a longer follow-up period are required to better de ne duration of drug effect over time.
Achieving the goal of primary prevention of viral wheezing both in healthy children and, even more, in patients with a positive clinical history for viral wheezing is still unsolved. In this scenario, PDT, thanks to its immune-modulatory effects, could be an innovative strategy, especially at our days sadly burdened by a new coronavirus (2019-nCoV) that also a icts the pediatric population, resulting commonly in viral respiratory infection [18]. In this regard, our ndings not only suggest the therapeutic effectiveness of PDT but also its preventive effectiveness, as assessed by the evidence that, after 3-months follow-up period, a decrease in the number of patients taking ICS and SABA, in the number of patients requiring ED visits and hospitalization was recorded.
The initiating and perpetuating factors of wheezing are multiple and their interactive processes are based on the interplay between genetic and environmental exposure which, in turn, in uence the development of the immune system. Therefore, whether PDT affects signi cantly the immune response of children with wheezing, it cannot change or modulate other causes of wheezing, such as genetic predisposition and environmental exposure. On the other hand, the behaviour of the PDT as a symptomatic drug is in line with its properties; acting as an immunomodulator agent, PDT can modify the immune response until it is administered, and, although it cannot change the natural history of the disease, it can decrease the severity disease as assessed by a signi cant reduction in the number of patients taking ICS and SABA as well as in the number of patients requiring ED visits and hospitalizations. Thus, although PDT does not appear as a de nitive treatment of wheezing our ndings evidence that it can be considered as a valid and adjunctive therapeutic approach in treating wheezing.
Lastly, compared to the previous clinical studies [16,17], we reported interesting ndings on the safety pro le of PDT and we showed that this immunomodulatory agent does not cause adverse effects neither at short-nor long-term.

Conclusion
Wheezing is characterized by signi cant morbidity and represents a very common health problem in the rst years of life for abnormal use of drugs, increased health care services and economic costs, due to ED visits and hospitalizations [1]. Our pilot study highlighted the ability of PDT to modify the course of the disease during the period of treatment but, rstly to date, maintaining its clinical effect over three months after treatment suspension, counteracting the recurrence of disease.
Furthermore, PDT seems able to stabilize the severity disease, although the enrolled patients were not receiving PDT and they were still in winter virus season, the number of patients taking concomitant drugs (ICS and SABA) and the number of patients requiring ED visits and hospitalizations were maintained over time. Further studies are required to con rm this pilot study and future investigations with a longer followup period will better de ne the duration of drug effect over time. Interestingly, in light of our encouraging ndings, we suggest that PDT administration might prove useful also in treating multi-trigger wheezing, often requiring a more complex therapeutic management.

Declarations
Ethics approval and consent to participate Institutional Review Board of University of Catania approved the study. A written informed consent was obtained from the parents and informed assent from the children and adolescents. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors have no con icts of interest to disclose that could be perceived as prejudicing the impartiality of the research reported. Funding The authors did not receive any funding for the research. Valeas, s.p.s. Italia (Milano, Italy) covers the article-processing charge. Authors' contributions SL designed the study. SM wrote the initial draft of the manuscript. SL performed a critical revision of the manuscript and offered precious technical advice on how the study might be improved. GFP, MP, FM, AG enrolled the children and contributed to the data collection and follow-up. GFP reviewed and edit the manuscript. All authors provided substantial contributions to the conception or design of the work, or the acquisition, analysis, or interpretation of data for the paper, revised the manuscript for important intellectual content, approved the nal version, and agreed to be accountable for all aspects of the work. Acknowledgments We would like to thank all the participants and their families who took part in this research.