**4. Discussion**

An investigational medication is defined as a drug and/or formulation that has been approved for clinical testing by either the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA), but has not gained marketing authorisation yet [101,102]. Over the last five years, results from 19 Phase I and II RCTs on investigational agents for the treatment of asthma reported data from sixteen classes of investigational agents. Specifically, these investigational drugs included AZD1402, BI 443651, CHF6366, CJM112, depemokimab, dexpramipexole, ecleralimab, etokimab, GB001, itepekimab, melrilimab, PC945, REGN1908-1909, remibrutinib, TD-8236, velsecorat, indacaterol acetate, and a probiotic. Overall, the quality of the studies was good, although often data were not published in full text articles; thus, scarce information was available to adequately perform the RoB assessment.

The investigational anti-IL-4Rα inhibitor AZD1402, the anti-IL-5 mAb depemokimab, the anti-IL-17A mAb CJM112, the anti-TSLP mAb ecleralimab, the ENaC inhibitor BI 443651, the MABA CHF6366, and the anti-Fel d 1 mAb REGN1908-1909 were proven effective in the treatment of asthma, although data almost exclusively regarded the assessment of lung function, and thus did not allow conclusions regarding symptoms control and the secondary endpoints of this systematic review. The effectiveness of the LABA indacaterol was confirmed even when delivered using the formulation with maleate salt, which demonstrated an effect that was comparable to the currently marketed indacaterol acetate on FEV1, PEF, and rescue medication use reduction. Among the investigational anti-IL-33 mAbs, only itepekimab, but not etokimab and melrilimab, effectively improved asthma outcomes compared to PCB, but generally there was no further improvement observed when itepekimab was combined with dupilumab. Treatment with the SGRM velsecorat was generally superior to PCB when administered at higher doses.

Overall, investigational agents did not show superiority to active controls, with the exception of itepekimab, which significantly reduced BEC compared to dupilumab monotherapy, and velsecorat, which induced a significantly greater improvement in FEV1 vs. PCB compared to that produced by FF vs. PCB.

The main efficacy outcome assessed by the RCTs included in this systematic review was FEV1. In this respect, BI 443651, depemokimab, ecleralimab, indacaterol maleate, itepekimab, REGN1908-1909, and velsecorat produced a statistically significant improvement in lung function compared to PCB, thus representing promising add-on therapies for asthma in the future. It is also worth mentioning the synthetic amino-benzothiazole dexpramipexole, which was found to markedly reduce BEC across all the administered doses in patients with moderate to severe eosinophilic asthma, despite no significant improvement in lung function, relative to PCB [28,29].

The anti-IL-33 mAbs etokimab and melrilimab, the DP2 antagonist GB001, the selective BTK inhibitor remibrutinib, the pan-JAK inhibitor TD-8236, and the antifungal triazole PC945 induced an effect on lung function that was similar to PCB.

Although FEV1 is generally recognised by the research community and regulatory agencies to be a suitable variable for airflow obstruction assessment [103], it is not the most relevant endpoint for testing investigational anti-inflammatory agents, including the DP2 antagonist GB001 and the pan-JAK inhibitor TD-8236, particularly for short-term assessment. Thus, for such treatments, other efficacy endpoints should be considered in future studies. Although probiotics utilised in dietary supplements reside in a sub-category under the general umbrella term of "foods" rather than drugs, according to both the FDA [104] and the European Food Safety Authority (EFSA) [105], the probiotic Probiotical® investigated in a Phase II RCT (NCT03341403) [44,45] for uncontrolled severe asthma was included in this systematic review and was treated as investigational agent. The hypothesis

of such RCT [44,45] was that Probiotical® could have an impact in asthmatic patients who were not optimally controlled, reducing the local and systemic inflammatory state and then improving QoL and asthma control [44,45]. This hypothesis was also supported by the evidence that certain probiotic strains have anti-inflammatory and immunomodulatory effects in pre-clinical models of asthma [106,107] and RCTs of adult asthma [108,109]. Interestingly, although dietary supplements are not subjected to the pre-market approval requirement for drugs, an investigational new drug application must be submitted to the FDA if the clinical investigation is intended to evaluate whether a dietary supplement is useful in diagnosing, curing, mitigating, treating, or preventing a disease, under the Code of Federal Regulations Part 312 [110]. In contrast, in the EU, there is still no specific regulation covering probiotics, pre-biotics, synbiotics, or postbiotics, but as suggested by The International Scientific Association of Probiotics and Prebiotics consensus statement, the definition of such products requires a health benefit; thus, it is expected that the use of any of these terms would require a health claim approval [111].

In any case, the daily administration of Probiotical® showed some improvement in sputum eosinophil count after 3 months of therapy, but in agreement with the current scientific evidence, the use of probiotics as adjuvant therapy for asthma is not yet conclusive [112]. Three meta-analyses carried out to explore the potential effects of probiotics in preventing allergic diseases and asthma led to conflicting outcomes due to a high degree of heterogeneity among the studies, mostly concerning the design, the characteristics of included patients, the analysed variables, and the used probiotic strains [113–115].

The assessment of efficacy outcomes reported by the RCTs included in this systematic review indicates that not only were some of the investigational agents superior to PCB from a statistical point of view, but they also elicited clinically relevant effects compared to PCB in asthmatic patients, as reported in Table 2. As a matter of fact, itepekimab 300 mg Q2W, indacaterol maleate 150 μg QD, and velsecorat 720 μg QD overcame the Minimal Clinical Important Difference (MCID) [103] threshold for trough FEV1 or risk of asthma exacerbation. Interestingly, velsecorat 720 μg QD, itepekimab 300 mg Q2W, and itepekimab 300 mg Q2W + dupilumab 300 mg Q2W were borderline to reach the MCID threshold for ACQ or AQLQ. Indeed, these promising results need to be confirmed by Phase III studies.

A main limitation of this systematic review is that most of the included studies (11 RCT, 57.9%) had a registry record on ClinicalTrials.gov and/or EU Clinical Trial Register but no associated publication, and thus, sponsors and principal investigators are exclusively responsible for the scientific accuracy of the provided results, which may be inconsistent across all the provided studies.

Additionally, findings for three RCT of the included studies were retrieved from grey literature which were not formally and rigorously peer reviewed, and thus should be carefully interpreted due to potential publication bias [116].

There is a strong pharmacological need to look beyond current therapeutic strategies and consider further promising biological drugs for asthma that are under development and for which results have not been posted on clinical trial registries and are not available in current literature.

Asthma remission is a complex condition that can be clinically defined as a sustained absence of symptoms, optimisation or stabilisation of lung function, and no use of OCS for exacerbation treatment [117], but controversy remains regarding the threshold of each item used to assess the asthma remission itself [118]. Although these terms do not necessarily imply the absence of airway pathology, a recent point of view suggested that asthma remission may be an achievable goal, at least in asthmatic patients with the T2 phenotype [117].

In conclusion, novel investigational agents, such as biologics, may have the potential to promote disease modification. Clearly, further larger studies are needed to confirm positive results from Phase I and II RCTs. So far, most of the investigated therapies have been evaluated as add-on options to current treatment, but it would be extremely advantageous for new therapies to be effective enough to replace current pharmaceutical options in order to simplify regimens of administration.

*Biomedicines* **2022**, *10*, 2330

**Table 2.** Clinical effect of investigational agents currently evaluated in Phase I and II RCTs for the treatment of asthma compared to PCB on efficacy outcomes for which the MCID values are currently available. The investigational agents reported in this table also elicited statistically significant improvement vs. PCB (*<sup>p</sup>* < 0.05).


long-acting β2-adrenoceptor agonist; mAb: monoclonal antibody; MCID: Minimal Clinical Important Difference; NA: not available; PCB: placebo; PEF: peak expiratory flow; Q2W: once every2weeks;QD:*quaquedie*,oncedaily;RCT:randomisedcontrolledtrial;SGRM:selectiveglucocorticoidreceptormodulator;TSLP:thymicstromallymphopoietin.

**Author Contributions:** All the authors (L.C., M.A., A.F., E.P., B.L.R., P.R. and A.C.) made substantial contributions to the conception or design of the work; the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; or have drafted the work or substantively revised it; all the authors (L.C., M.A., A.F., E.P., B.L.R., P.R. and A.C.) approved the submitted version; all the authors (L.C., M.A., A.F., E.P., B.L.R., P.R. and A.C.) agreed to be personally accountable for their own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and documented in the literature. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the article.

**Conflicts of Interest:** L.C. reports grants and personal fees from Boehringer Ingelheim, grants and personal fees from Novartis, nonfinancial support from AstraZeneca, grants from Chiesi Farmaceutici, grants from Almirall, personal fees from ABC Farmaceutici, personal fees from Edmond Pharma, grants and personal fees from Zambon, personal fees from Verona Pharma, personal fees from Ockham Biotech. M.A. has no conflicts of interest to declare. A.F. has no conflicts of interest to declare. E.P. has no conflicts of interest to declare. B.L.R. has no conflicts of interest to declare. P.R. reports grants and personal fees from Boehringer Ingelheim, grants and personal fees from Novartis, personal fees from AstraZeneca, grants and personal fees from Chiesi Farmaceutici, grants and personal fees from Almirall, grants from Zambon, personal fees from Biofutura, personal fees from GlaxoSmithKline, personal fees from Menarini, and personal fees from Mundipharma. A.C. received grants from Menarini and Astra Zeneca and a personal fee from Chiesi.
