**1. Introduction**

HHT is a rare but ubiquitous hereditary vascular disease, with an estimated prevalence of 1/5000 to 1/8000. The ENG (endoglin) and ACVRL1 encoding ALK1 (activin receptor-like kinase 1) genes are responsible for 90% of cases of HHT [1]. These genes both intervene in the BMP9/ALK1/ENG/SMAD pathway in endothelial cells, and it has been hypothesized that HHT is related to disequilibrium in the angiogenic balance, resulting from an increase in the factors involved in the activation phase and a decrease in those involved in the maturation phase of angiogenesis [2].

The recognized manifestations of HHT are all due to abnormalities in vascular structure. Lesions may be cutaneous and/or mucosal telangiectases or visceral arteriovenous malformations (AVMs) in the lungs, liver, and central nervous system [3]. Telangiectases and AVMs vary widely between individuals and even within the same family. Nosebleeds are the most frequent complication in HHT and may occur as often as several times per day. They are spontaneous, very variable in time and from one patient to another, but recurrent in 90% of patients. They can be associated with severe anemia in 2–10% of patients, and blood transfusions are required sometimes or regularly (every 2 or 3 weeks) in 2–5% of patients [4]. These nosebleeds thus significantly reduce quality of life [5].

The incomplete and transient efficacy of nasal surgical therapies has inspired a new search for adjuvant medical treatments which would greatly diminish daily iron loss [6]. For this reason, anti-angiogenic treatments such as intra-venous anti-VEGF treatment (bevacizumab) and thalidomide have been evaluated in clinical studies [7,8], but their use is limited to severe forms of the disease. Furthermore, local bevacizumab administration (nasal spray) recently evaluated in 2 phase 2 studies was not efficient [9,10]. We thus decided to investigate the feasibility and efficacy on epistaxis in HHT of other known anti-angiogenic drugs with a possible nasal administration and absorption that would target and reactivate the altered BMP9/ALK1/ENG/SMAD pathway.

Based on our collaboration with Bailly's group, and on the results obtained by Ruiz et al. [11], working on the repositioning approach developed by screening the libraries of Food and Drug Administration approved drugs that could potentiate the BMP9 signaling response, we concluded that the most promising activating drug was tacrolimus, a potent activator of the BMP9-ALK1-BMPR2-Smad1/5/9 signaling cascade. How tacrolimus activates this pathway is still not completely understood. Tacrolimus (FK506) can bind to FKBP12 (FK-506-binding protein-12), a protein known to interact with the TGF-ß family type I receptors [12]. Tacrolimus binding to FKBP12 leads to FKBP12 dissociation from the type 1 receptors, which can then activate the Smad transcription factors. Alternatively, tacrolimus has also been reported to stimulate endoglin and ALK-1 expression in endothelial cells, and to enhance the TGF-β1/ALK1 signaling pathway and endothelial cell functions such as tubulogenesis and migration [13]. In parallel, preclinical models have shown that injections of tacrolimus decreased the number of retinal arteriovenous malformations induced by BMP9/10-immunodepletion in a mouse HHT model [11]. These results sugges<sup>t</sup> that the mechanism of action of FK506 involves a partial correction of endoglin and ALK1 haplosufficiency, and may therefore be an interesting drug for use in patients with HHT. Furthermore, improvement in epistaxis has been shown in HHT patients after a liver transplant [14], and it has been hypothesized that the immunosuppressive treatment (FK506) used to prevent rejection may have an anti-angiogenic effect.

Topical nasal administration of tacrolimus may be an easy-to-use and non-invasive treatment. In addition, tacrolimus ointment is available on the market as a treatment for eczema and can therefore readily be used for nasal administration. Its tolerability on mucosae has been evaluated in the treatment of chronic plaque psoriasis and oral lichen planus [15–18]. Transient burning sensations have been described in tacrolimus patient groups, but no serious adverse e ffects necessitating stopping treatment have been recorded. Furthermore, none of the patients showed any abnormality in hematological or biochemical parameters. Data from healthy human subjects indicate that there is little or no systemic exposure to tacrolimus following repeated topical application of tacrolimus ointment. Several studies in infants [19,20] on the pharmacokinetics of tacrolimus after first and repeated application showed minimal systemic exposure (less than 1 ng per mL in all cases) and there was no evidence of systemic accumulation. Further to administration on mucosae, the pharmacokinetics were evaluated in two clinical trials and no systemic absorption was detected [16,18].

For all these reasons, tacrolimus ointment was a good candidate treatment for HHT. Our objective was to evaluate the e fficacy on the duration of nosebleeds of tacrolimus nasal ointment in patients with hereditary hemorrhagic telangiectasia complicated by nosebleeds.

#### **2. Materials and Methods**

#### *2.1. Trial Design and Treatment*

The study was a prospective, phase II multicenter, randomized study, ratio 1:1, carried out in a double-blind setting. It was approved by the local research ethics committee and by the French Medical Products Agency (ANSM) in March 2017. Written informed consent was obtained from all patients in accordance with national regulations. The trial was conducted in accordance with the principles of the Declaration of Helsinki [21] and Good Clinical Practice guidelines. All the authors were involved in designing or conducting the study, and preparing the manuscript, including the decision to submit it for publication. This trial was registered with the ClinicalTrials.gov Identifier #NCT03152019.

The nasal ointment was self-administered by patients, twice daily, for 6 weeks. About 0.1 g of product was to be administered in each nostril. The tube was gently squeezed to extract an amount roughly equivalent to the size of the head of a cotton swab. The ointment was then introduced into each nostril with a cotton swab, and extended into the nostril with a cotton swab or/and by external pressure on the nostril.

Marketed 30 g tubes of Protopic ® 0.1% (Léo Pharma, Voisins le Bretonneux, France) were used for this study. For the placebo, the manufacturing and filling were managed by an external pharmaceutical laboratory with GMP accreditation. The placebo formulation was similar to the active ointment; it contained all the ingredients except the active one: tacrolimus. The product was provided in strictly identical tubes. The same masked label was placed on both batches in order to respect the blind.
