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Research ArticleDrug Discovery and Translational Medicine

The Antiangiogenic Insulin Receptor Substrate-1 Antisense Oligonucleotide Aganirsen Impairs AU-Rich mRNA Stability by Reducing 14-3-3β–Tristetraprolin Protein Complex, Reducing Inflammation and Psoriatic Lesion Size in Patients

Sylvie Colin, Bernadette Darné, Amin Kadi, Antoine Ferry, Maryline Favier, Corinne Lesaffre, Jean-Pascal Conduzorgues, Salman Al-Mahmood and Nejib Doss
Journal of Pharmacology and Experimental Therapeutics April 2014, 349 (1) 107-117; DOI: https://doi.org/10.1124/jpet.113.209346

Abstract

Increased inflammation and aberrant angiogenesis underlie psoriasis. Here, we report that the inhibition of insulin receptor substrate-1 (IRS-1) expression with aganirsen resulted in a dose-dependent reduction (P < 0.0001) in IRS-1 protein in the cytoplasm, while IRS-1 protein remained quantitatively unchanged in the perinuclear environment. Aganirsen induced a dose-dependent increase in serine-phosphorylated IRS-1 in the soluble perinuclear-nuclear fraction, inducing IRS-1–14-3-3β protein association (P < 0.001), thereby impairing 14-3-3β–tristetraprolin protein complex and AU-rich mRNA’s stability (P < 0.001). Accordingly, aganirsen inhibited (P < 0.001) in vitro the expression of interleukin-8 (IL-8), IL-12, IL-22, and tumor necrosis factor alpha (TNFα), four inflammatory mediators containing mRNA with AU-rich regions. To demonstrate the clinical relevance of this pathway, we tested the efficacy of aganirsen by topical application in a pilot, double-blind, randomized, dose-ranging study in 12 psoriatic human patients. After 6 weeks of treatment, least square mean differences with placebo were −38.9% (95% confidence interval, −75.8 to −2.0%) and −37.4% (−74.3 to −0.5%) at the doses of 0.86 and 1.72 mg/g, respectively. Lesion size reduction was associated with reduced expression of IRS-1 (P < 0.01), TNFα (P < 0.0001), and vascular endothelial growth factor (P < 0.01); reduced keratinocyte proliferation (P < 0.01); and the restoration (P < 0.02) of normal levels of infiltrating CD4+ and CD3+ lymphocytes in psoriatic skin lesions. These results suggest that aganirsen is a first-in-class of a new generation of antiangiogenic medicines combining anti-inflammatory activities. Aganirsen-induced downregulation of inflammatory mediators characterized by AU-rich mRNA likely underlies its beneficial clinical outcome in psoriasis. These results justify further large-scale clinical studies to establish the dose of aganirsen and its long-term efficacy in psoriasis.

Introduction

Psoriasis is a common chronic disease occurring in ∼2% of the population in Western countries (Sabat et al., 2007; Nestle et al., 2009). Extensive work has established that increased inflammation, aberrant angiogenesis, and vascular remodeling as well as keratinocyte hyperproliferation are involved in the pathogenesis of psoriasis (Heidenreich et al., 2009).

T cell–mediated immune response to an as-yet-unknown autoantigen seems to play a key role in the pathogenesis of psoriasis. This deregulated immune response is primarily driven by CD4+ T cells with a T(h)1 and/or T(h)17 phenotype (Nograles et al., 2008; Nestle et al., 2009; Coimbra et al., 2012) and by the production of tumor necrosis factor alpha (TNFα) (Orlinick and Chao, 1998; Caldarola et al., 2009). A significant increase in the development of the microvasculature (angiogenesis) has been observed in psoriatic lesions compared with healthy skin (Heidenreich et al., 2009), especially in the early phase of the development of the lesions (Henno et al., 2010). Vascular endothelial growth factor (VEGF), which has a central role in angiogenesis, is upregulated in psoriasis (Detmar et al., 1994), and its levels of expression represent a good indicator of active psoriatic arthritis (Heidenreich et al., 2009).

Conventional systemic therapies for psoriasis, such as methotrexate, cyclosporin A, retinoids, or psoralen and UV A therapies, can result in long-term toxicity and may not be effective. The development of novel therapies targeting inflammatory factors including TNFα, interleukin-12 (IL-12), and IL-22 now provide new and efficient treatment options (Weger, 2010). Nonetheless, because both angiogenesis and inflammation are involved in the development of psoriasis, we speculated that aganirsen (GS-101) could be efficient in psoriatic patients. Aganirsen is an antisense oligonucleotide that inhibits the expression of insulin receptor substrate-1 (IRS-1) and has antiangiogenic activities, including inhibition of both VEGF and IL-1β expression (Andrieu-Soler et al., 2005; Al-Mahmood et al., 2009; Cloutier et al., 2012). In this study, we show that, by inhibiting IRS-1 expression in the cytoplasmic compartment, aganirsen impaired 14-3-3β–tristetraprolin protein (TTP) complex formation, leading to the inhibition of the expression of several cytokines with AU-rich mRNA, including IL-8, IL-12, IL-22, and TNFα. We tested the efficacy of aganirsen by a 6-week topical application in patients with chronic mild-to-moderate plaque psoriasis and showed that aganirsen reduced plaque area. This was associated with a reduction in the expression of VEGF and TNFα protein, as well as a normalization of the levels of CD4+ and CD3+ lymphocytes and reduced keratinocyte proliferation in psoriatic skin lesion biopsies. These data strongly support that the combined antiangiogenic and anti-inflammatory properties of aganirsen synergized to improve the clinical status of the patients.

Materials and Methods

Real-Time Reverse-Transcription Polymerase Chain Reaction Assay.

The human microvascular endothelial cell line (hEC) was provided by Dr. E. W. Ades (Centers for Disease Control and Prevention, Atlanta, GA), who established this line by transfecting human dermal endothelial cells with SV40A gene product and large T antigen (Ades et al., 1992). Cells were cultured in EGM-2-MV medium (Lonza, Levallois, France). After exposure to various concentrations of aganirsen (0–10 μM) or vehicle for 24 hours, hEC (5 × 105 cells/ml) total mRNAs were isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Hoerd, France). RNA yields and purity were assessed by spectrophotometric analysis. The real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed as described previously (Al-Mahmood et al., 2009). In brief, 0.5 μg of total RNA was reverse-transcribed with random hexamer primers and Moloney murine leukemia virus (200 U; Invitrogen, Carlsbad, CA), and the synthesized cDNA was used immediately for real-time PCR amplification using the DNA-binding dye SYBR Green I for the detection of PCR products and the following primers: TNFα (sense, 5′-GCTGCAGCACATTATAATACAGAGA-3′; antisense, 5′-GGTGTTTGTCGCGACTCC-3′); IL-8 (sense, 5′-AGTGGACCACACTGCGCCAAC-3′; antisense, 5′-CCACAACCCTCTGCACCCAGT-3′); IL-12 (sense, 5′-GAATGCAAAGCTTCTGATGGA-3′; antisense, 5′-GTGGCACAGTCTCACTGTTGA-3′); IL-22 (sense, 5′-CCCTCAATCTGATAGGTTCCAG-3′; antisense, 5′-GCAGGTCATCACCTTCAATATG-3′); and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (sense, 5′-TGAAGGTCGGAGTCAACGGA-3′; antisense, 5′-CATTGATGACAAGCTTCCCG-3′). The real-time PCR reactions were carried out with the DNA Light Cycler 480 (Roche Diagnostics, Meylan, France). The results were quantified using the equation: CopyTF/CopyGAPDH = 2C(t)GAPDH − C(t)TF, in which CopyTF is the number of copy of the targeted fragment gene and CopyGAPDH is the number of copy of GAPDH gene fragment used as internal control. All PCR products were analyzed by electrophoresis on 1.5% agarose gel, visualized with ethidium bromide, and analyzed using the Genesnap 6.00.26 software (Syngene, Frederick, MD). Densitometric analysis was performed using GeneTools Analysis Software version 3.02.00 (Syngene).

Subcellular Protein Fractioning and Protein Quantification.

Subcellular protein fractioning was realized using the Thermo Scientific subcellular protein fractionation kit for cultured cells (Product No. 78840; Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. The fractionation kit permitted the separation and preparation of five fractions: membrane, cytoplasmic, soluble perinuclear-nuclear (SPN), chromatin-bound, and cytoskeletal protein fractions from hECs. IRS-1 protein was only detected in the cytoplasmic and the SPN fractions. The protein content of the different fractions was measured by Bradford’s method and adjusted. For purity control within the subfractions, equivalent amounts of proteins were resolved by SDS-PAGE, followed by the transfer of proteins to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with 5% defatted milk solution in phosphate-buffered saline (PBS)–0.5% Tween for 1 hour, followed by two washes and immunoblotting with an anti–epidermal growth factor receptor monoclonal antibody (mAb) (clone D38B1, rabbit mAb; Cell Signaling Technology Inc., Danvers, MA) as plasma membrane marker; an anti–heat shock protein p90 (cytoplasmic fraction marker) (clone C45G5, rabbit mAb; Cell Signaling Technology, Inc.), an anti–histone deacetylase 2 mAb (rabbit mAb; Cell Signaling Technology, Inc.), and an anti–transcription factor Sp1 mAb as markers of the SPN fraction; an anti-vimentin mAb (clone D21H3, rabbit mAb; Cell Signaling Technology, Inc.) as a marker of the cytoskeletal fraction; and an anti–histone 3 mAb as a marker of the chromatin-bound insoluble nuclear fraction. The results of the purity controls of the obtained fractions are shown in Supplemental Fig. 1.

Serum-deprived hECs were incubated with different concentrations of aganirsen or scramble oligonucleotide at 37°C under 5% CO2 for 6 hours. After three washes with ice-cold PBS, cells were suspended with the protein extraction buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, and 1 μM phenylmethylsulfonyl fluoride]. The protein content was measured by Bradford. IRS-1 concentration in the extracts was determined by the Path-Scan Total IRS-1 Sandwich enzyme-linked immunosorbent assay (ELISA) kit (Cell Signaling Technology, Inc.) according to the manufacturer's instructions. The data were collected from four separate experiments performed in duplicate and expressed relative to control cells (vehicle).

To confirm the results obtained with the Path-Scan Total IRS-1 Sandwich ELISA kit, equal volumes of the adjusted cell extracts were also resolved by SDS-PAGE, followed by the transfer of proteins to a PVDF membrane. The membrane was incubated with 5% defatted milk solution in PBS–0.5% Tween for 1 hour, followed by two washes and immunoblotting with either an anti–IRS-1–horseradish peroxidase (HRP) conjugate (1:300 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti–GAPDH-HRP conjugate (Santa Cruz Biotechnology, Inc.), goat anti-TNFα (1:200 dilution; reference sc52746; Santa Cruz Biotechnology, Inc.), and anti–goat-HRP conjugate (1:20,000 dilution; reference sc2302; Santa Cruz Biotechnology, Inc.). Proteins were then monitored by ECLplus (GE Healthcare, Chalfont St. Giles, UK).

For the quantification of IL-8, IL-12, IL-22, and TNFα in the culture medium, hECs in culture medium without serum were incubated with different concentrations of aganirsen or scramble oligonucleotide at 37°C under 5% CO2 for 24 hours. Culture medium was recovered and used to quantify TNFα, IL-8, IL-12, and IL-22 using human TNF sandwich ELISA kit (reference 900-k25; Peprotech, Neuilly-sur-Seine, France) and human IL8, IL-12, and IL-22 sandwich ELISA kits (reference 900-k18, 900-K96, and 900-K246; Peprotech), respectively, according to the manufacturer's instructions.

Immunoprecipitation.

hECs in culture medium without serum were incubated with different concentrations of aganirsen or scramble oligonucleotide at 37°C under 5% CO2 for 6 hours. The cells were washed twice with cold PBS and lysed with 1% Triton X-100 buffer for 1 hour at 4°C. The protein 14-3-3 was immunoprecipitated by adding 4 μg of anti–14-3-3β mAb (reference sc628; Santa Cruz Biotechnology, Inc.), and the immune complexes were harvested with protein G-Sepharose beads (Santa Cruz Biotechnology, Inc.), washed with lysis buffer, and resolved in SDS-PAGE; and proteins were transferred to PVDF membrane and revealed with the indicated antibody.

Pilot Clinical Study.

The clinical study was approved by the institutional ethics committee and was conducted in accordance with Good Clinical Practices including International Conference of Harmonization Guidelines and all applicable local laws and regulatory requirements. It was designed as a double-blind, randomized, placebo-controlled, dose-ranging, Latin-square-design, single-center study from the Department of Dermatology of the Military Hospital of Tunis (see study protocol and results in Supplemental Data). It was set up to evaluate the safety and the efficacy of two doses of aganirsen versus placebo in 12 patients with chronic mild-to-moderate plaque psoriasis (Feldman and Krueger, 2005). Each patient had three psoriasis plaques (including two refractory lesions; refractory areas were elbow and knee). Each lesion was treated either by a low dose (0.86 mg/g) or a high dose (1.72 mg/g) of aganirsen or the ointment alone [placebo; 60% paraffin oil and 40% white Vaseline (v/v)]. At the inclusion and randomization visit (visit 1), the investigator selected for treatment at least two discoids or plaques from two refractory sites (elbow and knee) representative of the disease and a third plaque in a refractory or not refractory site. At visit 1 (inclusion in the study) and at visit 2 (3 weeks postinclusion), 75 tubes were delivered, i.e., 25 tubes of each type (A, B, and C). The investigator completed a form that was given to the patient together with the ointment tubes explaining what type (A, B, and C) of ointment tube must be used for each plaque. The order of plaques corresponded to the order recorded in the case report form. “A” and “B” types were assigned to refractory sites only. “A” ointment tubes were used for the first plaque, “B” ointment tubes were used for the second plaque, and “C” ointment tubes were used for the third plaque. The same one type of ointment tube was always used to treat a given psoriasis plaque throughout the study for a given patient.

Each index psoriasis plaque was photographed at inclusion before starting the treatment and every 3 weeks. A study nurse was trained to take these photos according to standards specifically developed for this study.

Population of the Pilot Clinical Study.

Before starting the pilot clinical study, the percutaneous absorption of aganirsen was studied quantitatively ex vivo on human dermatome skin biopsies mounted in Franz diffusion cells. Results showed that single topical application of aganirsen ointment resulted in a therapeutic concentration in both the epidermis and the dermis (Supplemental Fig. 2, A and B). No change in protocol of the pilot clinical study occurred. The main characteristics of the included patients were summarized in Table 1.

View this table:
TABLE 1

Main characteristics of the patients included in the pilot clinical study

Immunohistochemistry Analysis.

We collected three types of biopsies from three patients randomly selected at the end of the trial: healthy skin; placebo-treated psoriatic skin; and aganirsen (0.86 mg/g)-treated psoriatic skin. Tissue sections were stored at −80°C. For immunolabeling, they were thawed at room temperature, followed by incubation with a 3% bovine serum albumin solution in PBS for 1 hour at room temperature. Then they were labeled with the indicated mAb diluted to 1/100 in PBS–3% bovine serum albumin. The antibodies were incubated for 1 to 2 hours for the anti-TNFα mAb (mouse anti-human TNFα; clone 52B83; reference sc-52746; CliniSciences, Montrouge, France) and anti-VEGF mAb (clone C-1; Santa Cruz Biotechnology, Inc.), overnight at 4°C for anti-human CD3 polyclonal antibody (reference 14-0038-82; Affymetrix eBioscience, Santa Clara, CA) and anti-human CD4 polyclonal antibody (reference 14-0048-82; Affymetrix eBioscience). Tissue sections were then washed with PBS at room temperature and incubated with the horse anti-mouse IgG (H+L)-Fluorescein conjugate (reference FI-2000; CliniSciences) diluted to 1/100 in PBS for 1 hour. Following washes with PBS, tissue sections were mounted in aqueous medium (Vectashield; reference H-1500; Vector Laboratories, Burlingame, CA). The labeled tissue sections were examined with a Leica DMR fluorescence microscope equipped with a DC300F camera for image acquisition. The quantification of the labeling was performed using ImageJ software from NIH Image, and staining data were normalized and presented as % labeling area per lesion area. For each labeling, the n corresponds to the number of tissue sections analyzed. Immunohistochemistry procedure and analysis were repeated three times in independent series of experiments using the same tissue sections. This measure was made blinded to treatment.

Study Endpoints.

The primary efficacy endpoint was the percentage evolution of area of plaque size between baseline and week 6 as measured on photographs. This measure was made blinded to treatment. This efficacy assessment relied on a millimetric surface scale measurement of target lesions (pictures). All lesions within the scale square were measured. The sum of all areas of all lesions within the square was used. Therefore, the primary efficacy parameter was defined as:Embedded ImageMajor secondary efficacy endpoints were the percentage change in area after 3 weeks from baseline and change after 3 and 6 weeks from baseline in Total Sign Score (TSS) on physical examination and in Physician’s Global Assessment (PGA). Safety assessments were made at all visits by investigating adverse events, serious adverse events, and routine hematologic and laboratory values.

Statistical Analysis.

Primary and secondary efficacy analyses were based on the intent-to-treat population. The intent-to-treat population included all randomized patients. Safety analyses were based on the dataset comprising all randomized patients who received at least one dose of study treatment. All statistical tests were two-sided at the 5% significance level. The power level of the study was not considered given the small sample size. Normality of areas of target lesions, percentage changes in area of target lesions, and lesion diameter variables were checked visually and with the Kolmogorov-Smirnov test and Anderson-Darling test (Anderson and Darling, 1954). All parameters related to psoriatic lesion data were summarized by treatment group (placebo, 0.86 mg/g, and 1.72 mg/g) and target lesions. Continuous parameters, such as primary efficacy, were compared using analysis of variance. All data were analyzed using the SAS software version 9.2.

Results

Aganirsen Reduced IRS-1 Protein Expression in the Cytoplasmic Fraction, But Not in the SPN Fraction.

Aganirsen dose-dependently inhibited IRS-1 expression in hECs (Fig. 1, A and B) and human keratinocytes (Fig. 1, C and D). Subcellular fractionation of hECs permitted the separation of five fractions: membrane, cytoplasmic, SPN, chromatin-bound, and cytoskeletal protein. IRS-1 protein was only detected in the cytoplasmic and the SPN fractions. Closer examination of IRS-1 protein expression revealed that aganirsen inhibited IRS-1 protein expression in the cytoplasmic fraction only (Fig. 1E) and not in the SPN fraction (Fig. 1F). Analysis of the serine phosphorylation state of IRS-1 protein (pIRS-1Ser) revealed that pIRS-1Ser was undetectable in the cytoplasmic compartment (Fig. 1G); in contrast, there was a dose-dependent increase in pIRS-1Ser in the SPN fraction (Fig. 1H).

Fig. 1.
Fig. 1.

Aganirsen reduced IRS-1 protein expression in the cytoplasmic compartment but not in the SPN. (A) Equivalent amounts of protein extracts from hECs incubated with increasing concentrations of aganirsen were used to quantify IRS-1 protein by Western blot (upper). Membranes were stripped and reprobed with anti-GAPDH mAb to control for equal loading (lower). (B) The same hEC protein extracts as in A were used to quantify IRS-1 protein by ELISA sandwich assay. (C) Equivalent amounts of protein extracts from human keratinocytes incubated with increasing concentrations of aganirsen were used to quantify IRS-1 protein by Western blot (upper). Membranes were stripped and reprobed with anti-GAPDH mAb to control for equal loading (lower). (D) The same human keratinocyte protein extracts as in C were used to quantify IRS-1 protein by ELISA sandwich assay. (E) Equivalent amounts of the cytoplasmic fractions from hECs incubated with increasing concentrations of aganirsen were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti–IRS-1 mAb (upper). The membranes were stripped and reprobed with anti-GAPDH mAb to control for equal loading (lower). (F) Equivalent amounts of SPN fractions from hECs incubated with increasing concentrations of aganirsen were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with anti–pIRS-1 mAb (upper). The same membranes were stripped and reprobed with anti-actin mAb to control for equal loading (lower). (G) Equivalent amounts of the cytoplasmic fraction from hECs incubated with increasing concentrations of aganirsen were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with anti–pIRS-1 mAb. (H) Equivalent amounts of SPN fractions from hECs incubated with increasing concentrations of aganirsen were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with anti–pIRS-1 mAb. **P < 0.001; ***P < 0.0001 versus vehicle-treated group (0.9% NaCl). Scramble oligonucleotide (10 μM).

Aganirsen Impairs 14-3-3β–TTP Complex, Leading to the Inhibition of TNFα Expression.

In the SPN fraction, the increased serine phosphorylation of IRS-1 (Fig. 1H) paralleled the increased association of IRS-1 with the protein 14-3-3β. Increasing amounts of IRS-1 coimmunoprecipitated with the protein 14-3-3β (Fig. 2A); this was accompanied with decreasing quantities of the zinc finger protein TTP coimmunoprecipitated with 14-3-3β (Fig. 2B). These results suggest that the association 14-3-3β–IRS-1 impaired the formation of the complex 14-3-3β–TTP. TTP is a hyperphosphorylated protein that destabilizes mRNAs by binding to their AU-rich elements (Blackshear et al., 2003; Cao et al., 2003). The mRNA of many cytokines contains AU-rich elements, including those of TNFα, IL-8, IL-12, and IL-22 (Cao et al., 2003). The scramble oligonucleotide had no effect on the membrane-bound TNFα in hECs (Fig. 2D). In contrast, there was a dose-dependent decrease in membrane-bound TNFα in hECs incubated with aganirsen (Fig. 2D). This dose-dependent inhibition of TNFα expression by aganirsen was confirmed by the measurement of the secreted TNFα protein in the culture medium (Fig. 2F) and by the measurement of TNFα transcripts by quantitative RT-PCR (Fig. 2G). In addition, aganirsen led to a dose-dependent inhibition of IL-8 expression at both the translational (Fig. 2H) and the transcriptional levels (Fig. 2I), of IL-12 expression at both the transcriptional (Fig. 3A) and the translational levels (Fig. 3B), and of IL-22 expression at both the transcriptional (Fig. 3C) and the translational levels (Fig. 3D).

Fig. 2.
Fig. 2.

Aganirsen impairs 14-3-3β–TTP complex leading to the inhibition of both IL-8 and TNFα expression. (A) hECs incubated with increasing concentrations of aganirsen were lysed and proteins were immunoprecipitated with anti–14-3-3β mAb. Equivalent amounts of the immunoprecipitates were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti–IRS-1 mAb. (B) The same equivalent amounts of immunoprecipitates as in A were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-TTP mAb. (C) The membranes used in A were stripped and reprobed with anti–14-3-3β mAb to control for equal loading in A and B. (D) hECs incubated with increasing concentrations of aganirsen were lysed and equivalent amounts of proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with anti-TNFα mAb to reveal the membrane-bound TNFα. (E) The PVDF membranes used in (D) were stripped and reprobed with anti-actin mAb to control for equal loading in D. (F) hECs were incubated with increasing concentrations of aganirsen, and TNFα mRNA was measured by quantitative RT-PCR and expressed as mean ± S.E.M. (n = 4). (G) hECs in serum-deprived culture medium were incubated overnight with increasing concentrations of aganirsen, and the culture supernatants were collected to quantify secreted TNFα proteins by ELISA sandwich assay. Results are expressed as picograms of TNFα per milliliter of culture supernatant and presented as mean ± S.E.M. (n = 4). (H) hECs were incubated with increasing concentrations of aganirsen, and IL-8 mRNA was measured by quantitative RT-PCR and expressed as mean ± S.E.M. (n = 4). (I) hECs in serum-deprived culture medium were incubated overnight with increasing concentrations of aganirsen, and the culture supernatants were collected to quantify secreted IL-8 protein by ELISA sandwich assay. Results were expressed as picograms of IL-8 per milliliter of culture supernatant and presented as mean ± S.E.M. (n = 4). **P < 0.001; ***P < 0.0001 versus vehicle-treated group (0.9% NaCl). Scr, scramble oligonucleotide (10 μM).

Fig. 3.
Fig. 3.

Aganirsen inhibits both IL-12 and IL-22 expression. (A) hECs were incubated with increasing concentrations of aganirsen, and IL-12 mRNA was measured by quantitative RT-PCR and expressed as mean ± S.E.M. (n = 4). (B) hECs in serum-deprived culture medium were incubated overnight with increasing concentrations of aganirsen, and the culture supernatant was collected to quantify secreted IL-12 protein by ELISA sandwich assay. Results were expressed as picograms of IL-12 per milliliter of culture supernatant and presented as mean ± S.E.M. (n = 4). (C) hECs were incubated with increasing concentrations of aganirsen, and IL-22 mRNA was measured by quantitative RT-PCR and expressed as mean ± S.E.M. (n = 4). (D) hECs in serum-deprived culture medium were incubated overnight with increasing concentrations of aganirsen, and the culture supernatant was collected to quantify secreted IL-22 protein by ELISA sandwich assay. Results were expressed as picograms of IL-22 per milliliter of culture supernatant and presented as mean ± S.E.M. (n = 4). **P < 0.001; ***P < 0.0001 versus vehicle-treated group (0.9% NaCl). Scr, scramble oligonucleotide (10 μM).

Efficacy of Aganirsen in the Treatment of Psoriatic Lesions.

Treatment of 12 patients with topical application of 0.86 mg/g or 1.72 mg/g aganirsen for 6 weeks led to a significant reduction (P < 0.05) in the area of the treated lesions compared with placebo (Fig. 4, A and B; Table 2). By contrast, a slight increase in the lesion area was observed (median, 22%) in the placebo-treated group. Least square mean differences with placebo were −38.9% (95% confidence interval, −75.8 to −2.0%) and −37.4% (−74.3 to −0.5%) for the 0.86 mg/g and 1.72 mg/g groups, respectively. A significant decrease in aganirsen-treated lesion size was observed at as early as 3 weeks (Fig. 4, A and B; Table 2) of treatment compared with placebo (P < 0.01). Least square mean differences with placebo were −37.3% (−62.7 to −11.9%) and −38.3% (−63.8 to −12.9%) for the 0.86 mg/g and 1.72 mg/g groups, respectively.

Fig. 4.
Fig. 4.

Topical application of aganirsen reduced psoriatic lesion area compared with placebo. (A) Treatment of 12 patients with topical application of 0.86 mg/g or 1.72 mg/g aganirsen led to a significant reduction at 3 (†P < 0.01) and 6 weeks (‡P < 0.05) in the treated lesions compared with placebo. Data are mean ± S.D. (B) Representative images of the evolution of psoriatic lesions of patients treated with aganirsen at 1.72 mg/g (upper), patients treated with aganirsen at 0.86 mg/g (middle), and patients treated with placebo (lower). Visit 1, visit 2, and visit 3 correspond to baseline, after 3 weeks, and after 6 weeks of treatment, respectively.

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TABLE 2

Study clinical endpoints

We found no difference in the evolution of the TSS score (P = 0.63 and P = 0.82 at weeks 3 and 6, respectively; Table 2) between the three groups. At baseline, TSS was a median of 6; at week 3, a median of 4; and at week 6, a median of 3 in the placebo group. In the 0.86 mg/g treated group, the median TSS score was 5 at both baseline and week 3 and 4 at week 6, whereas in the 1.72 mg/g treated group, the median TSS score was 6 at baseline, 4 at week 3, and 3 at week 6. Likewise, the PGA score remained stable over the 6-week period and was similar in the three groups (Table 2).

Safety of Aganirsen.

Safety analysis was carried out on the 12 patients. Aganirsen's safety was good, without any reported adverse effects. Patients reported no irritation, burning, or exudation. In each treatment group, mild itching was reported for one lesion at baseline, no itching was reported at 3 weeks, and a moderate itching for one lesion at 6 weeks.

Aganirsen Inhibits IRS-1 and VEGF Expression in Psoriatic Lesions of Patients.

Treatment with aganirsen (0.86 mg/g) reduced IRS-1 expression by 81% ± 12% (P = 0.0132; n = 22) in human psoriatic skin lesions relative to placebo-treated psoriatic skin lesions (Fig. 5A; Supplemental Fig. 3). No difference in IRS-1 expression was detected between biopsies of healthy skin and placebo-treated lesions (Fig. 5A). In psoriatic lesion biopsies, we found that VEGF labeling was lowered by 42% ± 8% (P = 0.0074) in 0.86 mg/g treated psoriatic lesion biopsies compared with placebo-treated pathologic skin lesions (Fig. 5B).

Fig. 5.
Fig. 5.

Topical application of aganirsen for 6 weeks decreased both IRS-1 and VEGF expression in psoriatic lesion biopsies. (A) Inhibition of IRS-1 expression (measured by immunofluorescence using an IRS-1 antibody) after daily topical treatment of 6 weeks with aganirsen (0.86 mg/g ointment) in psoriatic skin lesion biopsies from patients. Data are mean ± S.E.M. *P = 0.0008 versus placebo-treated psoriatic lesions. (B) Inhibition of VEGF expression after daily topical treatment of 6 weeks with aganirsen (0.86 mg/g ointment). Data are mean ± S.E.M. *P = 0.0074 versus placebo-treated psoriatic lesions. (C) Representative images of VEGF labeling in human skin biopsies using anti-VEGF mAb in healthy untreated skin (left), placebo-treated psoriatic lesions (middle), and aganirsen-treated psoriatic lesions (right). Negative controls (secondary antibody only) are presented in the top row of images, cell nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) in blue (second row of images), immunostaining of VEGF is in green (third row of images), and the merge images incorporate both nuclei and VEGF immunostaining-derived fluorescence (fourth row of images).

Effects of Aganirsen on TNFα Expression, Inflammatory Cell Recruitment, and Keratinocyte Proliferation in Psoriatic Lesions of Patients.

Treatment with aganirsen (0.86 mg/g) was also associated with a lower TNFα labeling by 77% ± 11% (P = 0.0047; n = 14) (Fig. 6A; Supplemental Fig. 4) in skin biopsies of psoriatic patients compared with placebo-treated psoriatic skin samples. Measurement of CD4+ labeling indicated that psoriatic skin contained 65% ± 21% more cells (P = 0.013; n = 12) than healthy skin. In contrast, CD4+ labeling in aganirsen (0.86 mg/g)–treated pathologic tissue samples was lower by 43% ± 6% (P = 0.035; n = 12) compared with placebo-treated pathologic tissues (Fig. 6B; Supplemental Fig. 5). Similar results were obtained with CD3+ labeling. The psoriatic skin contained 115% ± 40% more (P = 0.0089; n = 14) CD3+ labeling than healthy skin, confirming our precedent results with CD4+ cells and further supporting the role of inflammation in psoriasis. CD3+ labeling in aganirsen-treated pathologic skin biopsies was reduced (47% ± 6%; P = 0.0355) compared with placebo-treated pathologic tissues (Fig. 6C; Supplemental Fig. 6); we found CD3+ labeling to be similar between healthy skin tissues and aganirsen-treated skin tissues. Finally, after 6 weeks of treatment with aganirsen, expression of proliferation marker Ki-67 and cytokeratin 16 were lower by 59% ± 3% (P = 0.0132; n = 10) and by 46% ± 8% (P = 0.0265; n = 5), respectively, compared with placebo-treated lesions (Figs. 7, A and B, and 8, A and B). Taken together, these findings demonstrate that aganirsen has potent anti-inflammatory effects and inhibits both the infiltration of lymphocytes and the proliferation of keratinocytes in the psoriatic skin.

Fig. 6.
Fig. 6.

Topical application of aganirsen inhibits TNFα, CD4+, and CD3+ lymphocyte infiltration in psoriatic lesions. Twice-daily topical treatment of 6 weeks with aganirsen at 0.86 mg/g ointment inhibited TNFα expression (A) in psoriatic skin lesions and reduced the levels of infiltrated CD4+ (B) and CD3+ (C) lymphocytes compared with placebo-treated psoriatic lesions. Data are mean ± S.E.M. (A) *P = 0.047 versus pathologic placebo-treated lesions; ‡P = 0.007 versus healthy skin. (B) *P = 0.002 versus pathologic placebo-treated lesions; †P < 0.0001 versus healthy skin. (C) *P = 0.0355 versus pathologic placebo-treated lesions; †P = 0.0089 versus healthy skin.

Fig. 7.
Fig. 7.

Topical application of aganirsen inhibits keratinocyte proliferation in psoriatic lesions. (A) Twice-daily topical treatment of 6 weeks with aganirsen at 0.86 mg/g ointment inhibited keratinocyte proliferation as evidenced by the reduced expression of the keratinocyte proliferation marker Ki-67 in psoriatic lesions. Data are mean ± S.E.M. *P = 0.0022 versus pathologic placebo-treated lesions; †P = 0.021 versus healthy skin. (B) Representative images of Ki-67 labeling in human skin samples using anti–Ki-67 mAb in healthy nontreated skin (left) and psoriatic lesions either placebo-treated (middle) or aganirsen-treated (right). Negative controls (secondary antibody only) are presented in the top row of images, cell nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) in blue (second row of images), immunostaining of Ki-67 is in green (third row of images), and the merge images incorporate both nuclei and Ki-67 immunostaining-derived fluorescence (fourth row of images).

Fig. 8.
Fig. 8.

Topical application of aganirsen inhibits cytokeratin 16 (CK-16) expression in psoriatic lesions. (A) Twice-daily topical treatment of 6 weeks with aganirsen at 0.86 mg/g ointment inhibited keratinocyte proliferation as evidenced by the reduced expression of CK-16 in psoriatic lesions. Data are mean ± S.E.M. *P = 0.0265 versus pathologic placebo-treated lesions. (B) Representative images of CK-16 labeling in human skin samples using anti–CK-16 mAb in healthy nontreated skin (left) and psoriatic lesions either placebo-treated (middle) or aganirsen-treated (right). Negative controls (secondary antibody only) are presented in the top row of images, cell nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) in blue (second row of images), immunostaining of CK-16 is in green (third row of images), and the merge images incorporate both nuclei and CK-16 immunostaining-derived fluorescence (fourth row of images).

Discussion

This study demonstrates that aganirsen is the first in its class of a new generation of antiangiogenic medicines that combines both antiangiogenic and anti-inflammatory activities and could be an effective treatment of patients with psoriasis. This statement is supported by several pieces of direct evidence, including the following: 1) in vitro, aganirsen induced IRS-1–14-3-3β association, which impaired 14-3-3β–TTP complex, leading to the inhibition of the expression of cytokines with AU-rich mRNA, including IL-8, IL-12, IL-22, and TNFα; 2) in psoriatic patients, aganirsen inhibited the expression of both VEGF and TNFα, thereby reducing the infiltration of inflammatory CD4+ and CD3+ lymphocytes; and accordingly 3) aganirsen inhibited keratinocyte proliferation and consequently reduced psoriatic lesion area in patients.

Psoriasis is a chronic inflammatory disease in which many inflammatory cytokines, including IL-12, Il-17, IL-22, and TNFα, play important pathologic roles. Thus, the most desired therapy is one that targets all these inflammatory factors. The mRNAs of IL-12, IL-17, IL-22, and TNFα share one characteristic, containing AU-rich elements at their 3′-untranslated regions (Bak and Mikkelsen 2010; Khabar, 2010). TTP is an mRNA-binding protein with high binding specificity for the so-called class II AU-rich elements within the 3′-untranslated mRNAs (Blackshear et al., 2003; Cao et al., 2003); specific binding of TTP to the AU-rich elements results in the destabilization and the decay of the mRNAs (Carballo et al., 1998; Lai et al., 1999). TTP occurs in a complex with 14-3-3, preventing TTP from binding to and/or directing mRNA to the degradation machinery (Johnson et al., 2002; Zhao et al., 2011). Our results show that aganirsen-induced association of IRS-1 with 14-3-3β protein impairs 14-3-3β–TTP complex, revealing a novel regulatory role for IRS-1. Indeed, IRS-1 associates with many of the seven isoforms of 14-3-3 proteins (Ogihara et al., 1997; Kosaki et al., 1998; Xiang et al., 2002; Oriente et al., 2005); yet the functional consequence for these associations was unknown. Our results are therefore the first to demonstrate that by promoting the association of IRS-1 to 14-3-3β protein and impairing 14-3-3β–TTP complex, aganirsen inhibits the expression of IL-8, IL-12, IL-22, and TNFα at both the transcriptional and the translational levels. This is in line with the importance of the liberation of TTP from the 14-3-3–TTP complex, and with the role of TTP in the regulation of expression of AU-rich elements in the destabilization and the decay of mRNAs (Carballo et al., 1998; Lai et al., 1999). These results, combined with those showing that aganirsen inhibited VEGF and IL-1β expression (Andrieu-Soler et al., 2005; Al-Mahmood et al., 2009), identify aganirsen as a dual-target therapeutic agent that is highly desirable for the treatment of psoriasis.

Topical applications of aganirsen led to therapeutic concentrations in both epidermis and dermis of human skin (Supplemental Fig. 2, A and B). It has been suggested that the impaired barrier function of psoriatic lesions (Schittek, 2011) and the amphipathic nature of aganirsen (Cloutier et al., 2012) could facilitate uptake of antisense drugs in general and aganirsen in particular. In agreement, topical applications of aganirsen decreased psoriatic plaque area in both groups of patients (0.86 and 1.72 mg/g) compared with placebo. Furthermore, aganirsen had a rapid onset of action as illustrated by the significant decrease in lesion area after 3 weeks of treatment of −14 and −13% in the 0.86 mg/g and 1.72 mg/g treated groups, respectively. By contrast, the plaque area increased by ∼15% in the placebo-treated group over this period. We did not find any significant differences in the evolution of TSS and PGA scores after either 3 or 6 weeks of treatment. This is most likely related to the short term of the trial; at least 12–16 weeks of treatment is required to confirm drug efficacy against psoriatic lesions (Papp et al., 2012a,b). In addition, our study did not reveal a dose-dependent effect of aganirsen; both 0.86 and 1.72 mg/g led to a similar decrease in psoriatic lesion area. These results suggest that the two doses tested in this pilot study induced a maximal effect, at least within the time frame of the current protocol. Lower daily doses of aganirsen combined with a longer period of treatment will therefore have to be tested in future clinical studies. Importantly, aganirsen was well tolerated locally, and no serious adverse effects were observed in any group. Nonetheless, our study was neither large enough nor of sufficiently long duration to ascertain uncommon adverse effects.

Psoriasis is characterized by chronic inflammation and enhanced lymphocyte infiltration (Nestle et al., 2009; Coimbra et al., 2012). Consistently, psoriatic lesions contained more CD4+ and CD3+ labeling compared with biopsies of healthy skin. Lymphocyte recruitment requires blood vessels, and aberrant angiogenesis has been linked to psoriasis (Detmar et al., 1998; Sabat et al., 2007; Heidenreich et al., 2009). In agreement with the antiangiogenic property of aganirsen, our results showed that VEGF expression was significantly lower in psoriatic lesions treated with aganirsen compared with placebo-treated psoriatic lesions, although VEGF expression was not elevated in the latter compared with that in the healthy skin. In contrast, CD31+ and CD34+ labeling was increased in psoriatic lesions compared with the healthy skin, and this increase was normalized following the treatment with aganirsen. In aggregate, these results suggest that the anti-inflammatory activity of aganirsen rather than its antiangiogenic activity underlies the observed beneficial clinical outcome. VEGF, nonetheless, is chemoattractant (Sawano et al., 2001; Ferrara et al., 2003) and promotes lymphocyte rolling and adhesion in skin microvessels (Detmar et al., 1998). Consequently, we would like to suggest that the inhibition of VEGF expression by aganirsen contributes to limit the recruitment of immune cells within the psoriatic lesions. This would explain the important decrease in TNFα protein in aganirsen-treated psoriatic lesions, which is mostly produced by activated leukocytes (Orlinick and Chao, 1998).

Psoriasis is characterized by epidermal thickening and keratinocyte hyperproliferation, which is directly linked to the inflammation within the psoriatic lesion (Sabat et al., 2007; Nestle et al., 2009). The expression of VEGF and TNFα was reduced by aganirsen in psoriatic lesions compared with placebo-treated lesions and was associated with >50% inhibition of keratinocyte proliferation as measured by Ki-67 and cytokeratin 16 (CK-16) labeling. The loss of CK-16–associated fluorescence following a 6-week therapy is a crucial finding since CK-16 is an important marker predicting therapeutic outcome (Krueger et al., 1995). The potent inhibitory action of aganirsen on keratinocyte proliferation is most probably linked to its anti-inflammatory action through inhibition of the expression of VEGF, TNFα, IL-12, and IL-22.

Finally, the levels of VEGF and TNFα were elevated in the biopsies of healthy skin, confirming the global proinflammatory status of these patients (Sabat et al., 2007; Nestle et al., 2009; Weger, 2010). Many anti-inflammatory drugs targeting TNFα, IL-12, IL-17, and IL-23 have been approved or are in advanced development stage for the treatment of psoriasis (Coimbra et al., 2012; Papp et al., 2012b). However, unlike aganirsen, which is active topically, these orally administrated new drugs have been associated with numerous side effects. Nonetheless, these new biologics have been used in patients with moderate-to-severe forms of psoriasis, while aganirsen was used in mild-to-moderate forms of the disease (Psoriasis Area and Severity Index score ranged from 4–7) and therefore cannot be compared.

In conclusion, our results demonstrate that aganirsen inhibits the expression of cytokines whose mRNA contains AU-rich elements at their 3′-untranslated regions, including TNFα, IL−1β, IL-8, IL-12, and IL-22, known targets of both inflammation and angiogenesis. On the other hand, we confirmed that aganirsen is a potent inhibitor of VEGF expression, the only known growth factor that induces inflammation and promotes angiogenesis and lymphangiogenesis (Al-Mahmood et al., 2009; Koch et al., 2011; Ji, 2012). Despite the limitations of our trial (the small sample size and the short-term duration of the treatment), these results suggest that aganirsen is a dual antiangiogenic and anti-inflammatory agent that could represent an innovative safe and effective alternative for the treatment of psoriasis, a disease in need of new and safe therapeutic options.

Acknowledgments

The authors thank Dr. Ekat Kritikou, Dr. Eric Thorin, and Eric Viaud for help in the writing and editing of the manuscript, and Céline Steverlynck (research engineer, Gene Signal SAS) and Maud Bongaerts (technician, Gene Signal SAS) for expert technical assistance.

Authorship Contributions

Participated in research design: Ferry, Doss, Al-Mahmood.

Conducted experiments: Colin, Darné, Favier, Lesaffre, Kadi.

Contributed new reagents or analytic tools: Conduzorgues, Al-Mahmood.

Performed data analysis: Al-Mahmood.

Wrote or contributed to the writing of the manuscript: Al-Mahmood.

Footnotes

    • Received September 5, 2013.
    • Accepted February 6, 2014.

  • S.C., B.D., S.A.-M., and N.D. contributed equally to this work.

  • This study was supported by Gene Signal SAS.

  • J.-P.C. is a consultant in product formulation for Gene Signal. S.C. and S.A.-M. are cofounders of Gene Signal, have stock ownership, and are employees of Gene Signal. A.F. possesses stock ownership of Gene Signal. B.D., A.K., M.F., C.L., and N.D. declare no conflicts of interest.

  • dx.doi.org/10.1124/jpet.113.209346.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

Abbreviations

CK-16
cytokeratin 16
ELISA
enzyme-linked immunosorbent assay
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
hEC
human endothelial cell
HRP
horseradish peroxidase
IL
interleukin
IRS-1
insulin receptor substrate-1
mAb
monoclonal antibody
PBS
phosphate-buffered saline
PCR
polymerase chain reaction
PGA
Physician’s Global Assessment
pIRS-1
phosphorylated insulin receptor substrate-1
PVDF
polyvinylidene difluoride
RT-PCR
reverse-transcription polymerase chain reaction
SPN
soluble perinuclear-nuclear
TNFα
tumor necrosis factor alpha
TSS
Total Sign Score
TTP
tristetraprolin protein
VEGF
vascular endothelial growth factor

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Research ArticleDrug Discovery and Translational Medicine

Aganirsen Improves Clinical Signs of Psoriasis

Sylvie Colin, Bernadette Darné, Amin Kadi, Antoine Ferry, Maryline Favier, Corinne Lesaffre, Jean-Pascal Conduzorgues, Salman Al-Mahmood and Nejib Doss
Journal of Pharmacology and Experimental Therapeutics April 1, 2014, 349 (1) 107-117; DOI: https://doi.org/10.1124/jpet.113.209346
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