Effect of baseline disease severity on achievement of treatment target with apremilast: results from a pooled analysis.

BACKGROUND: Treating to absolute treatment targets rather than relative measures such as Psoriasis Area and Severity Index (PASI)-75 is emerging as an important clinical concept included in psoriasis guidelines and clinical practice. Achieving treatment targets is associated with achievement of long-term outcomes. OBJECTIVE: To evaluate the relationship between psoriasis severity, disease characteristics and achievement of PASI ≤2 with apremilast in a pooled analysis of the phase 3 ESTEEM 1 and 2 (NCT01194219 and NCT01232283), phase 3b LIBERATE (NCT01690299) and phase 4 UNVEIL (NCT02425826) clinical trials. METHODS: Pooled data from patients with moderate-to-severe plaque psoriasis randomized to apremilast 30 mg BID were analysed by baseline PASI quartiles (Q1: 2.4-13.1; Q2: 13.2-15.9; Q3: 16.0-20.0; Q4: 20.1-57.8). Assessments included PASI, Dermatology Life Quality Index (DLQI), Scalp Physician's Global Assessment (ScPGA; ScPGA ≥1) and target (worst) Nail Psoriasis Severity Index (NAPSI; NAPSI ≥1). RESULTS: Of 1062 patients, 963 had ScPGA ≥1 and 643 had NAPSI ≥1; 771 patients with baseline and Week 32 PASI assessments were included in analyses of Week 32 PASI target achievement. Rates of PASI ≤2 at Week 32 were greater in lower PASI quartiles (Q1: 43.5%; Q2: 31.2%; Q3: 26.8%; Q4: 18.4%). Most patients achieving PASI ≤2 target (83.6%) achieved DLQI ≤5 at Week 32; 59.3% of patients who did not achieve PASI ≤2 target achieved DLQI ≤5. At Week 32, mean improvements in ScPGA and NAPSI were similar with more moderate vs. more severe disease (ScPGA, range: 1.1-1.4; NAPSI, range: 1.6-2.5). In a subgroup analysis, achievement of PASI ≤2 target was higher in the lowest PASI quartile and with disease duration <5 years. CONCLUSIONS: Greater achievement of PASI ≤2 was observed in patients with more moderate vs. more severe skin disease. Apremilast may be particularly beneficial in more moderate disease early in the treatment paradigm.

Efficacy and safety of adalimumab when added to inadequate therapy for the treatment of psoriasis: results of PRIDE, an open-label, multicentre, phase IIIb study.

BACKGROUND: Adalimumab is an effective treatment for chronic plaque psoriasis. OBJECTIVE: To evaluate the safety and efficacy of adalimumab for psoriasis patients who did not adequately respond to prior psoriasis therapy. METHODS: PRIDE (an Open-Label Access PRogram to Evaluate the Safety and Effectiveness of Adalimumab When Added to InaDEquate Therapy for the Treatment of Psoriasis) was a multicentre, Phase IIIb study in Canada. Patients with active moderate-to-severe plaque psoriasis who failed to respond to, or were intolerant of, prior therapies received adalimumab 80 mg at Week 0 followed by adalimumab 40 mg every other week Weeks 1 through 23. The primary efficacy measure was PASI (Psoriasis Area Severity Index) 75 response at Week 16. Secondary efficacy measures included PASI 90/100 and percentage change from baseline PASI score. Adverse events (AEs) and serious AEs were recorded. RESULTS: A total of 203 patients were enrolled at 26 sites. Baseline characteristics were: male, 61.1%; mean age, 45.5 years; mean PASI score, 20.0; previous exposure to biologics, 38.4%. At Week 16, PASI 75/90/100 responses were achieved by 70.9%/49.3%/24.1% of patients, respectively. Mean percentage PASI score decrease from baseline to Week 16 was 79.5%. Mean percentage PASI improvement and response rates were maintained through Week 24. Nasopharyngitis and upper respiratory tract infection were the only AEs to occur in ≥5% of patients. Nine patients experienced serious AEs; four were considered possibly or probably related to adalimumab. CONCLUSION: Adalimumab was safe, well-tolerated and effective for treatment of active plaque psoriasis in patients who had not adequately responded to prior therapy.

Inflammatory damage following first-generation replication-defective adenovirus controlled by anti-LFA-1.

First-generation replication-defective adenoviruses have been reported to lead to transient reporter gene expression due to a specific immune reaction involving T and B lymphocytes. Some recent reports have also demonstrated the presence of a nonspecific inflammatory reaction involving macrophages and neutrophils after both intramuscular injections and viral vectors transduction. To further investigate this nonspecific inflammatory reaction, deltaE1/E3a adenoviruses were injected intramuscularly in immunocompetent mice. Some of these mice were treated with anti-LFA-1. The adenovirus-injected muscles showed abundant CD4+, CD8+, LFA-1+, and Mac-1+ cell infiltration 3 days after the deltaE1/E3a injection. The anti-LFA-1 monoclonal antibody was able to block the nonspecific inflammatory damage due mostly to neutrophils and macrophages. The anti-LFA-1 did not produce this effect by reducing the muscle infiltration by LFA-1+ cells. It may instead have blocked the direct interaction between LFA-1 and ICAM-1 thus preventing the damage produced by the respiratory burst of neutrophils. Blocking the resulting damage of this inflammatory reaction with anti-LFA-1 in animals also treated with FK506, a powerful immunosuppressant for gene therapy, largely increased the long-term transgene expression compared with mice only treated with FK506.

Prevention of immune reactions triggered by first-generation adenoviral vectors by monoclonal antibodies and CTLA4Ig.

The therapeutic potential of adenovirus-mediated gene transfer using first-generation vectors is severely limited by the fact that only transient expression is achievable in immunocompetent animals. The loss in transgene expression can be attributed at least in part to the appearance of detrimental immune responses directed toward vector and/or transgene-encoded determinants. FK506 and cyclosporin A both reduced these immune responses. These immunosuppressants, however, may induce many severe side effects during prolonged use. An alternative strategy has been developed to overcome these problems following in vivo transfection of muscles of adult immunocompetent mice with a delta E1/E3a adenoviral vector encoding a beta-galactosidase (beta-Gal) expression cassette. YTS 177 (an anti-CD4 monoclonal antibody) as well as CTLA4Ig, a recombinant protein, partially controlled the immune responses. They were indeed able to reduce the muscle infiltration by CD4+ and CD8+ cells but they failed to repress the humoral response. Co-administration of YTS 191 (an anti-CD4), YTS 169 (an anti-CD8), and TIB 213 (an anti-CD11a) was, however, very efficient in blocking both cellular and humoral immune reactions. This combination of monoclonal antibodies allowed strong and stable transgene expression over 1 month. These data underline the potential of monoclonal antibodies as immunosuppressive adjunct treatment for adenovirus-mediated gene transfer.

FK506 immunosuppression to control the immune reactions triggered by first-generation adenovirus-mediated gene transfer.

Despite good initial success in vivo, gene transfer using first-generation replication-defective adenovirus has been reported to lead to transient reporter gene expression and to trigger inflammatory reactions in various organs and animal models. To gain more knowledge on this phenomenon, immune reactions were investigated following in vivo transfection of adult immunocompetent mouse muscle using a delta E1/E3a adenoviral vector encoding a beta-galactosidase (beta-Gal) expression cassette. Cellular and humoral immune reactions, and rejection of beta-Gal-positive muscle fibers, occurred within 3 weeks. The muscles showed massive infiltration by macrophages, natural killer cells, and CD8+ leukocytes. The mRNA levels of granzyme B and interferon-gamma were increased 6 days after vector injection, indicating that the infiltrating lymphocytes were activated. Antibodies were formed against the adenovirus group antigen and the beta-Gal gene product 2 weeks after construct injection. The immunosuppressant FK506, however, blocked the cellular infiltration and the humoral response and allowed strong, stable transgene expression over 1 month. These data emphasize the immune problems related to the use of delta E1/E3a adenoviruses as vectors for gene therapy, and they underline the potential of FK506 as an immunosuppressant adjunct treatment for adenovirus-mediated gene transfer.

Immunosuppression with monoclonal antibodies and CTLA4-Ig after myoblast transplantation in mice.

Various combinations of monoclonal antibodies specific for lymphocyte cell surface antigens and a recombinant molecule (CTLA4-Ig) were used to immunosuppress mice after transplantation of MHC-incompatible myoblasts. To assess the effectiveness of the immunosuppression, the donor myoblasts were obtained from a transgenic mouse (TnI LacZ1/29) containing a beta-galactosidase (beta-gal) reporter gene under the control of a muscle-specific promoter. No muscle fibers expressing beta-gal were observed 1 month after the myoblast transplantation, when the animals were not immunosuppressed or were treated with CTLA4-Ig alone. Approximately 50% of the muscle fibers expressed the beta-gal reporter gene 1 month after transplantation in mice treated with CTLA4-Ig combined with an anti-CD4 monoclonal antibody and in mice treated with a combination of anti-CD4, anti-CD8, and anti-lymphocyte function-associated antigen-1. The percentage of beta-gal-labeled muscle fibers increased to 94% when this combination of the three monoclonal antibodies was administrated weekly for 3 weeks. Although excellent graft survival rates were obtained 1 month after transplantation, reflecting an effective immunosuppression by these three treatments, no beta-gal-positive fibers were found 2 months after the transplantation, indicating the inability of these immunosuppressive agents to maintain long-term graft survival and induce tolerance to the myoblasts and muscle fibers of donor origin.

Increased granzyme B mRNA after alloincompatible myoblast transplantation.

Normal C57BL/10SnJ myoblasts were transplanted into the tibialis anterior of C57BL/10SnJ, C57BL/ScSn mdx, or BALB/c mice. These transplantations allowed us to investigate the immune response not only against MHC but also against dystrophin introduced in the dystrophic muscles by such transplantations. Recently, our group reported following myoblast transplantations cellular infiltration of the host muscle by class II MHC cells, macrophages, and lymphocytes expressing CD4 or CD8 and IL-2 receptors. In the present study, activation of these infiltrating lymphocytes was investigated by measuring the expression of granzyme B mRNA. We used reverse-transcriptase polymerase chain reaction to detect granzyme B mRNA at various intervals after myoblast transplantations. To standardize the results, the mRNA were reverse transcribed using an oligo (dt) so that beta-actin mRNA could also be amplified from the same cDNA preparation. Granzyme B mRNA was increased for at least 3 weeks after MHC alloincompatible grafts. The absence of increased granzyme B expression after allocompatible transplantation in mdx mice suggests that dystrophin is not sufficiently immunogenic to induce short term acute rejection. These results indicate that lymphocytes infiltrated in muscles injected with histoincompatible myoblasts are activated and sustain the requirement for an adequate immunosuppression after such transplantations.

Control of inflammatory damage by anti-LFA-1: increase success of myoblast transplantation.

Myoblast transplantation is a potential treatment for Duchenne Muscular Dystrophy. This article confirms by experiments in mice that one problem that has limited the success of clinical trials of this procedure is a rapid (within 3 days) inflammatory reaction which kills most of the injected myoblasts. The death of the transplanted myoblasts can be prevented by treating the host with a mAb against LFA-1. This led to a 27-fold increase in the number of muscle fibers expressing a reporter gene present in the donor myoblasts when the host is also adequately immunosuppressed with FK506. Therefore, both the nonspecific inflammatory reaction and the specific immune response should be adequately controlled following myoblast transplantation.

Resting lymphocyte kinase (Rlk/Txk) targets lymphoid adaptor SLP-76 in the cooperative activation of interleukin-2 transcription in T-cells.

Rlk/Txk is a T-cell-specific member of the Btk/Tec family of tyrosine kinases, whereas SLP-76 is a lymphoid adaptor that is essential for pre-TcR and mature TcR signaling. Although Rlk deficient T-cells show partial defects in T-cell proliferation, Rlk can complement ITK-/- cells with multiple defects in TcR initiated early events and interleukin (IL)-2 production. A key question is the nature of the target of Rlk responsible for bridging the TcR with the activation of IL-2 transcription. In this study, we identify a pathway in which Rlk phosphorylates SLP-76 leading to the phosphorylation of PLCgamma1, activation of ERKs, and the synergistic up-regulation of TcR-driven IL-2 NFAT/AP-1 transcription. Rlk phosphorylated the N-terminal region of SLP-76, a region that has been previously shown to serve as a target for ZAP-70. Loss of N-terminal YESP/YEPP sites of SLP-76 or the Rlk kinase activity attenuated cooperativity between Rlk and SLP-76. These observations support a model where the TcR can utilize Rlk (as well as ZAP-70) in the phosphorylation of key sites in SLP-76 leading to the up-regulation of Th1 preferred cytokine IL-2.

Lymphocyte infiltration following allo- and xenomyoblast transplantation in mdx mice.

Human and mouse (C57BL/10SnJ+/+) myoblasts were injected separately in the muscles of C57BL/10ScSn mdx/mdx mice. Mouse myoblasts (C57BL/10SnJ+/+) were also injected in normal mice (C57BL/10SnJ+/+ and BALB/c+/+). Some muscles that received a xenotransplantation (i.e., human myoblasts) were previously injected with a myotoxin, i.e., notexin. This treatment was not used for the allografts (i.e., mouse myoblasts). Human myoblast injections did not increase the number of dystrophin-positive cells above the background level due to backmutation. Moreover, the human myoblasts detected with an anti-HLA antibody decreased rapidly during the 6-week follow-up. The injection of normal mouse myoblasts in mdx mice did, however, increase the number of dystrophin-positive fibers. Moreover, numerous cells expressing mouse MHC class II, macrophages, granulocytes, neutrophils, natural killer cells, and a subset of T lymphocytes were detected by immunohistochemistry in cryostat sections of myoblast-injected muscles. These cells were present within 1 week of the myoblast injection in the muscle regions containing injected human or mouse myoblasts, and progressively decreased during the 6-week follow-up in the human myoblast transplantation. Lymphocyte infiltration reached a significant level following xeno- and alloincompatible transplantations. Antibodies against the human myoblasts and against alloincompatible myoblasts were also detected in the serum of the recipients. These results suggest that humoral and cellular immune reactions are responsible for the poor outcome of myoblast transplantation in mice and could be involved in failure of transplantation in Duchenne muscular dystrophy patients. These results indicate that adequate immunosuppression must be used in these patients.

Human myoblast transplantation in immunodeficient and immunosuppressed mice: evidence of rejection.

Normal human myoblasts were cloned and transplanted in the tibialis anterior of immunodeficient nude and SCID mice and in mdx mice under different immunosuppressive treatments (cyclosporine A, CsA; antilymphocyte serum, ALS) or not immunosuppressed. This permitted us to show the interaction of the immune system in the myoblast transplantation. The graft success was assessed by verifying signs of humoral and cellular immune reactions and the presence of dystrophin produced by the fusion of the donor myoblasts. This study showed that clones of human myoblasts were able to fuse and produce dystrophin in injected muscles of immunodeficient mice and mdx mice receiving an effective immunosuppressive treatment (i.e., ALS+CsA). However, the same pool of human myoblasts injected in mdx mice inadequately immunosuppressed (i.e., CsA alone or ALS alone) triggered an immune reaction and was rejected. Cells expressing CD4 and CD8 antigens were observed in the injected muscles of mice treated with CsA alone. Therefore, evidence of humoral and cellular rejection was observed following human myoblasts transplantation.

Quantification of normal dystrophin mRNA following myoblast transplantation in mdx mice.

A mutagenesis RT-PCR method was used to detect normal dystrophin mRNA following the injection of normal myoblasts in mdx mice using two immunosuppressors. A specific sequence of the dystrophin mRNA (257 bp) was amplified by RT-PCR from the muscle total RNA. MaeIII digestion of the amplified products allowed us to distinguish the normal messenger of dystrophin from the dystrophic one and to establish the percentage of normal and of dystrophic (mdx) dystrophin mRNA. Normal dystrophin mRNA was detected using this technique in mdx muscles transplanted with histocompatible normal myoblasts. For this type of transplantation, no significant difference in the percentage of normal dystrophin mRNA was observed between immunosuppressed mice and those not immunosuppressed. No normal dystrophin mRNA was, however, observed in mdx mice following histoincompatible normal myoblast transplantation without immunosuppression. When such transplantations were done in mice immunosuppressed with cyclosporine or FK-506, normal dystrophin mRNA accounted for 31% and 36% of the total dystrophin mRNA, respectively. In fact, one animal immunosuppressed with FK-506 expressed as much as 57% of normal dystrophin mRNA. These results thus show that FK-506 makes it possible to restore dystrophin expression to a level comparable to that observed in DMD carriers that are usually asymptomatic.

Very efficient myoblast allotransplantation in mice under FK506 immunosuppression.

Transgenic CD1 mice expressing beta-galactosidase were used as myoblast donors. The myoblasts were injected in normal or mdx muscles previously irradiated and injected with notexin. Twenty-eight days after myoblast transplantation, the percentage of muscle fibers beta-glactosidase-positive was low in mice not immunosuppressed but was high (80%) in those treated with FK506. In mdx mice, muscle fibers expressing beta-galactosidase were also dystrophin positive. Most of the mice not treated with FK506 produced antibodies against the donor myoblasts. These results indicate that FK506 is a very useful immunosuppressive drug for myoblast transplantation in mice. Irradiation and notexin injection used in our experiments are, however, not feasible in humans. Other manipulations capable of increasing the participation of donor myoblasts to regeneration will therefore have to be identified before new clinical trials are attempted.

Increased interferon-gamma mRNA expression following alloincompatible myoblast transplantation is inhibited by FK506.

Myoblasts from C57BL/10SnJ+/+ were transplanted in major histocompatibility complex (MHC)-compatible mice (i.e., C57BL10SnJ+/+ and C57BL/10SnSc mdx/mdx) and in MHC-noncompatible (BALB/c+/+) mice. The recipients were killed 1-21 days after transplantation. C57BL10SnJ+/+ myoblasts were also transplanted in a few BALB/c+/+ mice treated with FK506 and killed 7 days thereafter. Our results showed that after MHC-noncompatible transplantation, interferon-gamma (IFN-gamma) mRNA expression is increased from day 5 to day 21, indicating the presence of a cellular immune reaction. Short-term immunosuppressive treatment with FK506 inhibited the transcription of IFN-gamma mRNA compared with that in untreated mice. Myoblast-specific antibodies were also detected 2 and 3 weeks after MHC-incompatible transplantation, indicating that the cellular immune reaction, revealed by the increase in IFN-gamma, was followed by a humoral reaction.