Apremilast in refractory orogenital ulcers and other manifestations of Behçet's disease. National multicenter study of 51 cases in clinical practice.

Oral and/or genital aphthous ulcers are the most common symptoms of Behçet´s disease (BD), and are often refractory to conventional treatment. The inhibitor of phosphodiesterase-4 apremilast (APR) has demonstrated ecacy in the treatment of this manifestations. The objective of the present study was to assess the ecacy of APR in the management of refractory oral and/or genital ulcers in patients with BD. National multicenter open-label observational study on BD patients with recurrent oral and/or genital ulcers. In all cases orogenital ulcers were refractory to conventional therapy. APR was given and maintained at standard dose of 30 mg twice daily. The main outcome was the achievement of oral and/or genital ulcers remission. Ecacy of APR for other clinical manifestations was also evaluated.

Conclusion APR yielded a rapid and maintained improvement of refractory mucocutaneous ulcers of BD, even in patients refractory to several systemic drugs including biologic therapy.

Background
Behçet´s disease (BD) is a chronic systemic in ammatory disorder of unknown etiology included in the group of variable vessel vasculitis [1,2]. It is characterized by a wide range of heterogeneous clinical manifestations and the treatment depends mainly on the clinical severity and affected organs [3,4].
Major organ involvement such as ocular, neurologic, vascular and gastrointestinal disease often requires an aggressive approach, usually with immunosuppressive agents [5,6]. Although recurrent oral and/or genital ulcers are not life-threatening complications, they are one of the most characteristic features of BD. Moreover, they can be extremely painful and disabling, [7,8]. Several systemic therapeutic agents such as colchicine, glucocorticoids, conventional and biologic immunosuppressive drugs have been used for orogenital aphthous ulcers with contradictory and variable results [9]. Apremilast (APR) is an orally active small molecule which inhibits phosphodiesterase-4 (PDE-4). APR modulates intracellular in ammatory pathways decreasing proin ammatory and increasing antiin ammatory mediators [10,11]. This drug is included in the group of targeted synthetic diseasemodifying antirheumatic drugs (tsDMARDs). Although combination therapy with two biological diseasemodifying antirheumatic drugs (bDMARDs) is generally not recommended [12], APR may be used in monotherapy or combined with either conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) and/or bDMARDs.
Randomized clinical trials (RCTs) are conducted under highly standardized design with strict inclusion criteria, excluding some real-world patients and special situations to make the statistical assessment of e cacy and/or safety more e cient [13]. Because of that, features of RCTs may differ from those of clinical practice, which could in uence the results of the treatment [14]. Two randomized double-blinded phase II and III clinical trials have shown e cacy and safety of APR for oral ulcers of BD [15,16]. Based on these trials, the U. S. Food and Drug Administration (FDA) has recently approved APR for the treatment of oral ulcers associated with BD (www.fda.gov). However, in these trials, patients with active involvement of any major organ during the 12 months before recruitment, history of recurrent or chronic infections, latent tuberculosis or who had received biologic therapies were not included. Furthermore, patients were not allowed to receive concomitant medications indicated for the management of BD. Full information related to orogenital ulcers prior to APR onset was not available in these two trials. Moreover, follow-up was of only 28 weeks and the e cacy of APR for manifestations different from orogenital ulcers was not reported.
Taking into account all these considerations, the aim of the present study was to assess the e cacy of APR for orogenital ulcers, either combined or in monotherapy, in a National multicenter clinical practice study of BD patients with orogenital ulcers refractory to conventional treatment. Moreover, the e cacy of APR for other clinical manifestations was also evaluated.

Design and Enrollment Criteria
We performed a multicenter open-label observational study that encompassed 51 BD patients with refractory mucocutaneous ulcers. Besides topical treatment, oral colchicine, non-steroidal anti-in ammatory drugs (NSAIDs) and systemic glucocorticoids, patients had received at least one csDMARD and in most cases bDMARD before the onset of APR.
Patients were diagnosed with BD at the Rheumatology, Autoimmune Diseases or Dermatology Units of 20 referral Spanish hospitals. The study was approved by the Clinical Research Ethics Committee. APR was prescribed as an off-label indication and, therefore, written informed consent was also requested and obtained from all patients.
BD diagnosis was performed according to the International Study Group for BD (ISGBD) criteria reported in 1990 [17]. As indicated by the Spanish National Guidelines for bDMARDs and tsDMARDs in Rheumatology [18][19][20][21][22], infections as well as malignancies were ruled out before starting the treatment. APR was initiated using dose escalation until reaching a maintenance dose of 30 mg twice daily.

Data Collection
Data were gathered from the clinical records of the patients according to a speci c designed protocol that included clinical and laboratory data, diagnosis, pharmacological agents used for the treatment of BD, response to APR and development of side effects. Data were reviewed for con rmation and stored in a computerized database. To minimize entry error, all the data were double checked.

Outcome Variables, Clinical De nitions And Laboratory Data
The primary outcome variable was the e cacy of APR to achieve remission of oral and/or genital ulcers. For this purpose, we assessed remission and ares of oral and/or genital ulcers. Complete remission was considered as the disappearance of ulcers while partial remission was de ned as the reduction of at least 50% in the number of ulcers and/or a reduction in the number of ares. Flare was de ned as the recurrence of ulcers when complete remission was achieved for at least one month. Similar de nitions (complete remission, partial remission and are) were applied when we assessed the effect of APR other clinical manifestations.
We also assessed safety and retention rate of APR as well as the sparing glucocorticoid effect due to the use of this molecule.
Serum C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR), full blood cell count, liver and renal function tests were also analyzed. ESR values higher than 20 or 25 mm/1st hour for men or women, respectively, and those of serum CRP greater than 0.5 mg/dL were considered raised.
Outcome variables were recorded in most patients at baseline (APR onset) and in every visit at 1-2 weeks, 4 weeks, 3 months, 6 months, 12 months, 18 months and 24 months. These visits were performed in each individual center following a pre-established protocol agreed by the investigators of this collaborative study.
An additional subanalysis considering APR in monotherapy or combined with bDMARDs and csDMARDs was also performed.
Adverse events related to APR treatment were evaluated, recorded and stored in a speci c le designed for this purpose.

Statistical Analysis
Results were expressed as mean ± standard deviation (SD) for variables with a normal distribution, or as median and interquartile range (IQR) [25th -75th IQR] for those not normally distributed. The effect of APR was assessed on clinical symptoms, serum CRP and ESR values and on daily glucocorticoid dose required. Comparisons were performed at baseline, 1-2 weeks, 4 weeks, 3 months, 6 months, 12 months, 18 months and 24 months using the Wilcoxon´s signed rank test. In addition, clinical and laboratory data of last visit were also assessed. Statistical signi cance was considered as a p-value ≤ 0.05. Statistical analysis was performed with the STATISTICA software (StatSoft, Tulsa, OK, USA).

Results
Demographic and general data at apremilast onset A series of 51 patients (35 women/16 men) diagnosed with BD and treated with APR was evaluated. The mean ± SD age at APR onset was 44.7 ± 13.2 years. HLA-B51 was positive in 20 patients (39.2%), negative in 27 (52.9%) and data were not available in another 4 cases (7.9%). The median [IQR] time from the diagnosis of BD to APR onset was 48  months.
An additional subanalysis comparing the e cacy of APR in monotherapy vs APR combined with csDMARDs and/or bDMARDs was carried out. However, there were not statistically signi cant differences in baseline characteristics and outcome (See Supplementary Table 1, Additional File 1).

Outcomes Of Orogenital Ulcers And Other Clinical Manifestations
Forty-four of 49 patients with available data at week 2 (89.8%) experienced a rapid improvement of the orogenital ulcers. Maintained clinical improvement of orogenital manifestations was also observed in most cases ( Table 2). As shown in the Supplementary Table 2, Additional File 2, the outcome of the orogenital ulcers was similar in patients treated with APR in monotherapy to those in whom APR was used in combination with conventional or biologic DMARDs.

Discussion
The results from the present study indicate that in clinical practice APR yields a rapid and maintained improvement of BD´s refractory orogenital manifestations. This is of potential relevance since oral and genital ulcers are the most representative manifestations of BD [8,23,25].
Due to the different phenotypes of the disease and the lack of consensual standards of care, BD treatment remains to be a challenge. The use of therapies is in many cases based on a few randomized clinical trials, singular case reports or small case series [26,27]. European League Against Rheumatism (EULAR) group has published an update of recommendations for the management of BD depending on the domain(s) affected in each patient, providing a more individualized therapeutic approach [9].
Several therapeutic agents have been used for orogenital aphthous ulcers with variable results [28]. There is general agreement on the use of topical agents such as chlorhexidine, lidocaine gel and glucocorticoid preparations for oral mucosal involvement. Alpsoy et al. described effectiveness of sucralfate suspension for oral and genital ulcers [29]. Colchicine remains as the rst-line systemic agent used for orogenital features of BD because of its inhibition of neutrophil chemotaxis [26,30,31]. This drug has proved to be useful for the treatment of erythema nodosum, genital ulcers of women and arthritis. However, there is no full evidence on its e cacy in oral ulcers [26,[32][33][34][35]. Kaneko et al. [36] reported that minocycline can reduce the frequency of oral ulcers, erythema nodosum and papulopustular lesions in BD patients as well as the production of pro-in ammatory cytokines by BD-peripheral blood mononuclear cells stimulated with streptococcal antigen. AZA is another drug used to avoid the development of mucocutaneous lesions of BD [37]. Thalidomide has shown e cacy for the treatment of oral and genital ulcers and papulopustular lesions in patients with BD. Nevertheless, maintenance treatment is frequently required to prevent the development of recurrences [26,31,[38][39][40][41], which together with the possibility of the appearance of nodular lesions and worsening of erythema nodosum, as well as the serious adverse events that this drug can cause, limit its use. Cyclosporin is another agent relegated to the background, due to its adverse events [26,31]. Sharquie et al. showed that dapsone was effective for the treatment of mucocutaneous lesions of BD in a double blind, placebo-controlled clinical trial [42]. With respect to TNFi, etanercept is the only drug assessed in a randomized controlled clinical trial that proved e cacy to suppress many mucocutaneous features, leading to a decrease in the frequency of appearance of oral ulcers and papulopustular lesions [4,26]. There are also case reports of successful treatment of genital ulcers with adalimumab [26,43]. Interferon (IFN) α has been used in mucocutaneous lesions with contradictory results and a high rate of adverse events [44][45][46]. A few studies suggest that anakinra, secukinumab and ustekinumab may be useful in the treatment of orogenital ulcers of BD [47][48][49][50].
APR is an oral small molecule which inhibits PDE-4 and increases the levels of intracellular cyclic AMP, modulating several in ammatory pathways [10,11]. A randomized phase II trial that included 111 patients with BD showed that patients treated with APR had a signi cant reduction in the number of oral ulcers at 12 weeks [15]. However, this trial did not provide enough information on previous therapies and extra-mucocutaneous manifestations. A recent phase III trial has shown signi cant improvement of pain and number of oral ulcers in 104 patients treated with APR, resolution maintained over 12 weeks and in many cases also resolution of genital ulcers [16]. Because of that, the U.S. FDA has recently approved APR for BD ulcers (www.fda.gov). However, in these trials the patients with major organ involvement of BD or comorbidities were excluded and full information on former therapies before APR was not available. Also, the time of follow-up was not very long. Moreover, to the best of our knowledge, both trials used APR in monotherapy and they did not assess the effect of APR on extra-mucocutaneous manifestations of BD [15,16]. Thus, the strict criteria required for inclusion may constitute a limitation at the time of considering APR in real-life patients.
In our study, APR achieved a rapid and sustained response of mucocutaneous ulcers in patients refractory to several systemic drugs, including biologic therapy. Adverse events were mild and most of them well tolerated. According to our ndings, APR may be combined with either with csDMARDs and/or bDMARDs with acceptable safety pro le. Nevertheless, we are aware of potential limitations of our study, mainly due to its retrospective nature.

Conclusion
In conclusion, we report real life data showing that APR therapy is effective in highly refractory BD orogenital ulcers.

Declarations
Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of clinical research of Cantabria, Spain (2018.100). All patients gave their informed consent for inclusion in the study.

Consent for publication
This manuscript does not contain any individual person's data.
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
BA-M received grants/research supports from Kern Pharma, AbbVie, P zer, Celgene and GSK.
JLM-V received grants/research supports from AbbVie, P zer and Celgene.
GE received grants/research supports from Actelion, Janssen, GSK, Boehringer and Amgen.
TP-S had nothing to disclose.
MM-M received grants/research supports from P zer, Janssen, Stada and Sandoz.
ED-A had nothing to disclose.
MDG-A had nothing to disclose.
EM had nothing to disclose.
IC had nothing to disclose.
FS had nothing to disclose.
JC-A had consultation fees/participation in company sponsored speaker's bureau from Celgene.
IdlM had nothing to disclose.
FO-S had nothing to disclose.
JAR-I had nothing to disclose.
AP-G had nothing to disclose.
SH had nothing to disclose.
AO had consultation fees/participation in company sponsored speaker's bureau from Celgene.
AP-E had nothing to disclose.
CD had nothing to disclose.
JJA had nothing to disclose.
AY had nothing to disclose.
AMF had nothing to disclose.
JN had nothing to disclose.
IF had consultation fees/participation in company sponsored speaker's bureau from Celgene.
AIT had nothing to disclose.
SRY had nothing to disclose.
PT had nothing to disclose.
SO had consultation fees/participation in company sponsored speaker's bureau from Celgene.
IR received grants/research supports from Celgene as principal investigator of PREVAIL study on apremilast in psoriasic arthritis.
VC-R had consultation fees/participation in company sponsored speaker's bureau from Abbvie, Lilly, MSD, UCB Pharma and Celgene.
CG-V had nothing to disclose.
SC had nothing to disclose.
JLH had nothing to disclose.
MAG-G received grants/research supports from Abbott, MSD and Roche, and had consultation fees/participation in company sponsored speaker´s bureau from Abbott, P zer, Roche and MSD.
RB received grants/research supports from Abbott, MSD and Roche, and had consultation fees/participation in company sponsored speaker´s bureau from Abbott, P zer, Roche, Bristol-Myers, Janssen and MSD.

Funding
There was no funding for this study.
Authors' contributions BA-M, JL-MV, JL and RB have made substantial contributions to the conception and design of the work.
All authors have contributed to the acquisition, analysis and interpretation of the data and have drafted the work. JL, SC, MAG-G and RB have substantively revised the work. All authors have agreed to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work are appropriately investigated, resolved, and the resolution documented in the literature. Submitted version of the manuscript has been approved by all authors.