Standardized Dermatophagoides mix (D. pteronyssinus + D. farinae) in TBU/ml in liquid formulation for sublingual immunotherapy: Phase II, randomized dose determination clinical trial.

The primary objective of this study is to determine the most effective dose of a standardized allergen extract mix containing Dermatophagoides pteronyssinus and Dermatophagoides farinae, administered via sublingual route and standardized in TBU/mL, for immunotherapeutic treatment without compromising patient safety. This dose-finding evaluation is clinically relevant for optimizing the therapeutic efficacy of sublingual immunotherapy in patients with allergic rhinitis and rhinoconjunctivitis caused by dust mite allergy, while maintaining an acceptable safety profile.

The secondary objectives include:

• Assessment of rescue medication use in relation to the investigational product or placebo, excluding any rescue medication administered following the nasal provocation test or skin prick test.

• Evaluation of the safety of the investigational treatment by considering both systemic and local adverse reactions reported during the trial that are related to the investigational product or placebo, excluding adverse reactions resulting from the nasal provocation test or skin prick test.

• Evaluation of the evolution of specific IgE to Dermatophagoides pteronyssinus, Dermatophagoides farinae, and the major allergens Der p 1 and Der p 2, from baseline to the end of the study.

• Evaluation of Dermatophagoides-specific IgG4 levels at baseline prior to immunotherapy and at the end of the study.

• Evaluation of the evolution of skin prick test responses to Dermatophagoides pteronyssinus and Dermatophagoides farinae from baseline to the end of the study.

• Independent analysis of each variable included in the assessment of the nasal provocation test outcomes.

The sponsor did not provide information regarding the total number of participants enrolled in this clinical trial. The study population consisted of individuals aged **14 to 50 years**, with both **male and female subjects** included. Participants were diagnosed with **allergic rhinitis** with or without **conjunctivitis**, with or without associated **asthma**. For those with associated asthma at recruitment, **forced expiratory volume in one second (FEV₁)** was required to be greater than 70% of predicted and **forced vital capacity (FVC)** greater than 80% of predicted. All participants demonstrated positive **skin prick test** results to **Dermatophagoides pteronyssinus** and **Dermatophagoides farinae**, with a mean wheal diameter at least 5 mm greater than the negative control. Additionally, **specific IgE** levels to both dust mite species were required to be equal to or greater than 0.70 kU/L, and a positive **nasal provocation test** result at a maximum concentration of 1/10 was mandatory. Women of childbearing potential were required to provide confirmation of non-pregnancy at study entry through a negative urine pregnancy test. Participants had no previous or concomitant **allergen immunotherapy** with Dermatophagoides extract or any other allergen during the study period. The trial population was selected based on specific diagnostic criteria confirming **dust mite allergy** and appropriate respiratory function parameters.

This Phase II clinical trial evaluates the optimal dose of a standardized **allergenic extract** mixture containing **Dermatophagoides pteronyssinus** and **Dermatophagoides farinae** administered via **sublingual route** for **immunotherapy** treatment in patients with **allergic rhinitis** or **rhinoconjunctivitis** caused by **dust mite allergy**, with or without associated **asthma**. The study follows a **randomized**, **dose-determination** design to identify the most effective dose without compromising patient safety. The investigational products consist of sublingual spray solutions at concentrations of 1000 TBU/ml, 3000 TBU/ml, and 6000 TBU/ml, alongside a **placebo** containing **sodium chloride**. Additionally, a nasal provocation test product containing allergenic extract of Dermatophagoides pteronyssinus is utilized for diagnostic purposes. The maximum daily dose for the sublingual products is 0.2 ml, with a maximum total dose of 24 ml administered over a treatment period of 4 months. The maximum daily dose for the nasal provocation test is 0.8 ml, with a maximum total dose of 1.6 ml administered over 1 day.

The **primary endpoint** assesses individual response to the **nasal provocation test** by measuring changes in **Nasal Inspiratory Peak Flow** using a **rhinomanometer**, with a positive response defined as a decrease of ≥40%. Clinical assessment is performed using a 13-point symptom scoring scale. **Secondary endpoints** include evaluation of rescue medication use throughout the study, monitoring of **adverse reactions** during the follow-up period, analytical determination of serum markers including **specific IgE** to Dermatophagoides pteronyssinus, Dermatophagoides farinae, Der p1, and Der p2, as well as **IgG4** to Dermatophagoides at baseline and study completion. Additional assessments comprise evaluation of primary wheal size from **skin prick tests** with Dermatophagoides pteronyssinus and Dermatophagoides farinae at baseline and study end, and analysis of all individual variables from the nasal provocation test scoring scale.

Participant eligibility requires age between 14 and 50 years, diagnosis of allergic rhinitis with or without conjunctivitis, with or without associated asthma, and in patients with asthma, **FEV₁** must be >70% of predicted and **FVC** >80% of predicted. Positive skin prick test results to both Dermatophagoides pteronyssinus and Dermatophagoides farinae with mean wheal diameter at least 5 mm greater than negative control, performed within 12 months prior to inclusion, are required. Specific IgE to both allergens must be ≥0.70 kU/L, determined within 6 months prior to inclusion. A positive nasal provocation test result at a maximum concentration of 1/10 is mandatory. Signed **informed consent** is required for study participation. Women of childbearing potential must provide confirmation of non-pregnancy through negative **urine pregnancy test** at study entry, with an additional test performed at treatment completion. Participants must not have received previous or concomitant allergen immunotherapy with Dermatophagoides extract or any other allergen during the study period.

The estimated recruitment start date is September 1, 2025, with an estimated study completion date of February 2, 2026. The expected duration of participant involvement is 4 months for the treatment phase. The study protocol includes systematic evaluation of safety parameters throughout the treatment period, with continuous monitoring for adverse reactions. Early termination from the study may occur under conditions that compromise participant safety or protocol compliance, although specific termination criteria are determined according to standard clinical trial safety procedures and regulatory requirements.

The experimental medications in this clinical trial consist of three different concentrations of a standardized allergenic extract mixture containing **Dermatophagoides pteronyssinus** and **Dermatophagoides farinae**. These products are biological medicinal products classified as standardized allergen extracts. All three experimental formulations are presented as **sublingual spray, solution** and are administered via **sublingual use**. The three dose strengths evaluated are 1000 TBU/ml, 3000 TBU/ml, and 6000 TBU/ml. The maximum daily dose for each concentration is 0.2 ml, with a maximum total dose of 24 ml administered over a treatment period of 4 months. The products are manufactured by ASAC PHARMACEUTICAL INMUNOLOGY.

The **placebo** comparator used in this trial is formulated as a sublingual spray, solution containing **sodium chloride** as the active substance. This placebo product is administered via the same sublingual route as the experimental treatments to maintain blinding. The dosing regimen for the placebo matches that of the experimental products, with a maximum daily dose of 0.2 ml and a maximum total dose of 24 ml over a 4-month treatment period. The placebo formulation is designed to be indistinguishable from the active treatments in terms of appearance and administration method.

An auxiliary diagnostic product is utilized in this trial for **nasal provocation testing**. This product consists of an allergenic extract of mites from the species Dermatophagoides pteronyssinus, presented as a powder for solution for inhalation. It is administered via **nasal use** for diagnostic purposes. The maximum daily dose is 0.8 ml, with a maximum total dose of 1.6 ml administered over a period of 1 day. This product serves as a standardized challenge agent to assess allergic responses during the trial.

All investigational products in this trial are standardized in TBU/ml units to ensure consistency and reproducibility of dosing. The treatment duration for the sublingual immunotherapy products and placebo extends over 4 months, allowing for adequate assessment of dose-response relationships. Participant compliance monitoring is essential given the self-administered nature of sublingual immunotherapy and the requirement for daily dosing throughout the treatment period. The trial design incorporates randomization to compare the efficacy and safety profiles of the different dose concentrations against placebo.

Efficacy will be assessed through the individual response of each subject to the nasal provocation test. The primary efficacy parameter involves objective measurement of the change in Nasal Inspiratory Peak Flow using a rhinomanometer, with a positive response defined as a decrease of at least 40 percent. Additionally, clinical assessment will be performed based on the total sum of symptom scores on a 13-point scale. Secondary efficacy parameters include the assessment of rescue medication use throughout the study in relation to the investigational product or placebo. Analytical determinations of serum markers will be conducted at baseline and at the end of the study, including specific IgE to Dermatophagoides pteronyssinus, Dermatophagoides farinae, Der p1 and Der p2, as well as IgG4 to Dermatophagoides. The assessment of the primary wheal resulting from skin prick tests with Dermatophagoides pteronyssinus and Dermatophagoides farinae will be performed at baseline and at the end of the study, calculated as the mean of the two diameters. The group mean will be calculated based on the results of each enrolled subject to allow for comparative analysis between groups. Evaluation of all individual variables from the scoring scale used in the nasal provocation test will also be conducted. Adverse reactions occurring during the patient follow-up period will be evaluated throughout the study.