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Extended report
An open-label pilot study of infliximab therapy in diffuse cutaneous systemic sclerosis
  1. C P Denton1,
  2. M Engelhart2,
  3. N Tvede2,
  4. H Wilson1,
  5. K Khan1,
  6. X Shiwen1,
  7. P E Carreira3,
  8. F Diaz Gonzalez4,
  9. C M Black1,
  10. F H van den Hoogen5
  1. 1
    Royal Free Hospital, London, UK
  2. 2
    Herlev University Hospital and Rigshospitalet, Copenhagen, Denmark
  3. 3
    Hospital 12 de Octubre, Madrid, Spain
  4. 4
    University Hospital, Tenerife, Canary Islands
  5. 5
    University Hospital, Nijmegen, The Netherlands
  1. Correspondence to Professor C P Denton, Centre for Rheumatology, Royal Free Hospital, Pond Street, London NW3 2QG, UK; c.denton{at}medsch.ucl.ac.uk

Abstract

Aim: The safety and potential efficacy of a chimaeric anti-tumour necrosis factor alpha monoclonal antibody (infliximab) were examined in diffuse cutaneous systemic sclerosis (dcSSc).

Methods: A 26-week open-label pilot study in which 16 cases of dcSSc received five infusions of infliximab (5 mg/kg). Clinical assessment included skin sclerosis score, scleroderma health assessment questionnaire, self-reported functional score and physician global visual analogue scale. Collagen turnover, skin biopsy analysis and full safety evaluation were performed.

Results: There was no significant change in skin score at 26 weeks but a trend for lower modified Rodnan skin score at 22 weeks (OR 17, 95% CI 6 to 46) compared with peak value (OR 29, 95% CI 11 to 44; p = 0.10). Serum aminoterminal propeptide of type III collagen level was significantly lower at week 26 compared with baseline (p = 0.03). Secretion of type I collagen by dermal fibroblasts was reduced at 26 weeks compared with baseline (p = 0.02). There were no deaths during the study and no suspected unexpected serious adverse reactions. 21 serious adverse events (AE) occurred in seven subjects, mostly attributable to dcSSc. 127 distinct AE occurred in 16 subjects. Of these, 19 AE (15%) were probably or definitely related to infliximab treatment. Eight (50%) patients prematurely discontinued infliximab. Anti-infliximab antibodies developed during the study in five subjects and were significantly associated with suspected infusion reactions (p = 0.025).

Conclusion: In dcSSc infliximab did not show clear benefit at 26 weeks but was associated with clinical stabilisation and a fall in two laboratory markers of collagen synthesis. The frequency of suspected infusion reactions may warrant additional immunosuppression in any future studies in systemic sclerosis.

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Systemic sclerosis (scleroderma; SSc) is a multisystem connective tissue disease of unknown aetiology that results in fibrosis and vasculopathy. Treatment is difficult and previous studies have often failed to demonstrate beneficial effects on the skin or other organ-based pathologies using a variety of therapies including immunosuppressive agents, putative antifibrotic drugs, or highly specific targeted biological therapies.1,2 In other autoimmune rheumatic diseases therapeutic blockade of tumour necrosis factor alpha (TNFα), a proinflammatory cytokine with effects on multiple cell types, has proved remarkably effective.3 Therefore, for rheumatoid arthritis, ankylosing spondylitis and other rheumatic diseases anti-TNFα therapy is well established. It is also beneficial in some skin diseases, notably psoriasis4 and inflammatory bowel disease.5

The rationale for evaluating anti-TNFα treatment in SSc is based upon clinical overlap of SSc with other rheumatic diseases in which benefit is well established, such as inflammatory joint disease, and effectiveness in conditions such as Crohn’s disease, in which fibrosis and scar formation is triggered by chronic inflammation.6 In addition, experimental models of lung fibrosis have responded to TNFα antagonists, and there is evidence that a recombinant soluble TNFα receptor may be helpful in idiopathic pulmonary fibrosis.7 This progressive often fatal form of interstitial lung disease shares many similarities with lung fibrosis in SSc. The rationale for TNFα antagonism in SSc is further supported by the inflammatory nature of skin disease in the early stages of diffuse cutaneous systemic sclerosis (dcSSc) and the presence of a mononuclear cell infiltrate including monocytes in skin biopsy specimens.8

However, there is also some concern about using anti-TNFα in SSc. It has been shown that TNFα may downregulate the expression of key profibrotic mediators such as connective tissue growth factor,9 which are implicated in SSc pathogenesis.10 In addition, TNFα is a potent stimulus for proteolytic enzymes including collagenase, and antagonists may reduce the breakdown or remodelling of fibrous extracellular matrix,11 and a recent study suggested that TNFα may be antifibrotic in SSc.12 Concerns are also raised by clinical data suggesting that in heart failure13 anti-TNFα drugs are harmful and can aggravate some autoimmune diseases including multiple sclerosis.14 There are reports of systemic lupus erythematosus and other autoimmune disorders being triggered by TNFα antagonists.15 Together these concerns make a thorough safety evaluation of TNFα blockade in SSc imperative. There has already been limited use of TNFα antagonists in SSc. A study of 10 cases treated with etanercept suggested that this agent was tolerated and may be associated with clinical improvement.16 However, cases in this series were heterogeneous and improvements in skin score may have resulted from the well-reported spontaneous improvement that occurs in many cases of dcSSc between 12 and 36 months after disease onset. Another more recent series of cases of arthritis–scleroderma overlap syndrome treated with etanercept was encouraging.17 In the present study we have treated a cohort of dcSSc patients who have documented evidence of progressive skin disease using infliximab to assess safety and look for evidence of possible efficacy.

Methods

Study design

This was an open label study. Subjects entering the study received infliximab 5 mg/kg at weeks 0, 2, 6, 14 and 22. All patients gave full informed consent. The protocol and patient informed consent forms were approved by the responsible ethics committees at participating sites. Specific inclusion criteria for the study included fulfilment of the American College of Rheumatology preliminary classification criteria for scleroderma and designation to the diffuse cutaneous subset of SSc, as evidenced by skin sclerosis proximal to the elbows or knees and absence of the anticentromere autoantibody. Documented recent progression of skin sclerosis (ie, skin score increasing by more than 10% or increase of four skin score units over a maximum of 3 months) was mandatory. Specific exclusion criteria included evidence of infection or malignancy on a screening chest radiograph, the use of putative disease modifying drugs (d-penicillamine, methotrexate, azathioprine, mycophenolate mofetil) or other potential immunosuppressive drugs within 4 weeks of screening. All potential subjects underwent tuberculin skin testing and to exclude suspected latent or active tuberculosis. As a result of concerns about administration of infliximab in the context of significant cardiac involvement, patients with an estimated left ventricular ejection fraction of less than 50% on echocardiography were excluded. Similarly, to avoid treating patients with severe interstitial lung disease patients with lung function tests showing forced vital capacity less than 55% of predicted values were also excluded.

Clinical outcomes

The primary efficacy analysis was based upon a stabilisation or improvement in the modified Rodnan skin sclerosis score (MRSS) at 26 weeks compared with baseline. MRSS was assessed by a single observer for each patient. The 17-site modified MRSS was used, with each site assessed by global average integer score 0–3. Consistent with clinical trial guidelines,18 and to minimise interobserver variation, all investigators attended skin scoring standardisation sessions, as earlier work suggests that this reduces interindividual variability in skin scoring.19,20 Progression to end-organ severe involvement or an increase in the number of organ systems affected was considered a surrogate for disease deterioration.21 Clinical assessment of study subjects followed routine clinical practice and standardised clinical trial design by focusing on determining the extent of skin involvement and the presence of major visceral involvement. Renal function was determined by creatinine clearance. Pulmonary function testing was performed by standard techniques and included forced vital capacity, total lung capacity, forced expiratory volume in 1 s and diffusing capacity. All subjects completed the scleroderma health assessment questionnaire (HAQ), which comprised the disability index of the HAQ and patient global visual analogue scale (VAS) for severity assessments of overall SSc, Raynaud’s phenomenon, lung disease, digital ulcers and gastrointestinal disease.22 The scleroderma UK functional score was also completed. This is an 11-item self-reported questionnaire that has been shown in cross-sectional and longitudinal studies to correlate with the disability index of the health assessment questionnaire (HAQ-DI).23 A physician global assessment of overall SSc severity was also determined using a VAS.

Serum biomarker evaluation

Potential markers of the underlying immunological, vascular and fibroblast dysfunction in SSc were assessed.24 Serum samples were shipped frozen to the lead centre for analysis. Biomarkers of collagen turnover were evaluated in serum samples and skin biopsies. Values at baseline and after 6 months were compared. Serum aminoterminal propeptide (PIIINP) was measured by commercial radioimmunoassay (Behringwerke AG, Germany and Orion Diagnostica, Helsinki, Finland, respectively).25 A quantitative immunoassay was used to assess IL-2R levels (R&D Systems, Inc, USA),26 and as a marker of vascular dysfunction.27

Type I procollagen secretion by fibroblasts

Skin biopsies (4 mm2) were taken from clinically affected and clinically unaffected skin (usually the lower back). Repeat biopsies were taken from sites within 5 cm of the first procedure. Biopsy specimens were shipped in refrigerated sterile culture medium to the lead centre for analysis. Fibroblast cultures were established from dcSSc skin biopsies at baseline and the end of the study, taken from matched sites on the forearm. Culture supernatants were collected from confluent fibroblast cultures of cells in the third passage. Supernatant proteins were concentrated using ammonium sulphate precipitation and resuspension. Collagen was detected by using specific primary antibody directed against type I procollagen. After medium concentration equal loading volumes were analysed by Western blot.28 Densitometric quantification was performed using the gelplate computer program, with normalisation against glyceraldehyde-3-phosphate dehydrogenase in the cell layer lysate to control for cell number.

Immunostaining for TGFβ1

Paraffin embedded formalin fixed sections of the skin biopsies were compared. Involved and uninvolved skin and baseline or end of study staining intensity was compared. Staining intensity was assessed using semiquantitative assessment of epidermal and dermal staining by an experienced observer, blinded to sample identity during scoring. Ten randomly selected high power fields were thus scored for each biopsy. A specific primary antibody for transforming growth factor beta 1 (TGFβ1) was used. An alkaline phosphatase conjugated secondary antibody was used and an appropriately diluted isotype matched irrelevant control antibody was used in place of the primary antibody in order to exclude non-specific binding of the anti-TGFβ1 primary reagent.

Adverse events

Safety was assessed by summarising the incidence and type of adverse events (AE) up to week 48. All patients were included in the safety assessment. AE were recorded in the case record form. Special attention was paid to (reactivation of latent) tuberculosis and congestive heart failure.

Assessment of anti-infliximab antibody formation

The development of anti-infliximab antibodies was determined using a previously described assay system.29 Serum samples were compared at baseline and 26 weeks. The presence of infliximab in the 26-week samples was ascertained as this can confound interpretation of the anti-infliximab antibody results. Samples were designated as positive at 26 weeks if there was significant anti-infliximab activity that was not present at baseline.

Database and statistical analysis

All outcome analysis was performed according to intention to treat. Parametric and non-parametric testing was undertaken according to the distribution of the sample data. Statistical significance was defined by p⩽0.05.

Results

Study subjects

Sixteen patients were enrolled into the study. The demographic and clinical features are summarised in table 1. The mean age of the subjects at entry was 48 years (SD 13). The majority had early stage disease (mean duration 15.7 months (SD 19.9)) and all had documented progression of skin sclerosis at entry into the study. Disease was extensive in most cases, with mean MRSS at baseline of 24.9 (SD 9.5). The mean baseline HAQ-DI was 1.55 (SD 0.86) and the scleroderma functional score was 14.4 (SD 9.5). Seven subjects discontinued treatment during the study. All were due to suspected infusion reactions and these occurred at the third infusion (n  =  3) at 6 weeks, the fourth infusion (n  =  2) at 14 weeks and the fifth infusion (n  =  2) at 22 weeks.

Table 1

Patient characteristics

Safety and tolerability

An important goal of this study was to assess the safety and tolerability of infliximab in dcSSc. There were no deaths during the study or up to the 48-week safety assessment and no suspected unexpected serious adverse reactions. Twenty-one serious AE occurred in seven subjects and the majority were attributable to dcSSc. These included sclerodema renal crisis (new hypertension and renal impairment), infected digital ulcers, severe Raynaud’s, digital ischaemia, pyrexia and new-onset atrial fibrillation. One possible infusion reaction resulted in an unscheduled hospital admission and so was classified as a serious AE. Two serious AE were considered to be probably related to study medication. There were 127 distinct AE, occurring in 16 subjects. Of these, 19 AE (15%) were probably or definitely related to infliximab treatment. Eight subjects discontinued infliximab prematurely and details of these cases are provided in table 2.

Table 2

Summary of eight subjects discontinuing infliximab prematurely during study

An important observation concerning safety was the high frequency of possible infusion reactions during the study, occurring in seven of 16 subjects (44%) that may have reflected the absence of concurrent immunosuppression in this study. Many were mild or very mild and occurred between the third and fifth infusion. Nevertheless, study medication was stopped as they occurred within 10 minutes of starting infliximab administration and there was a low threshold for suspicion in a study exploring the use of this agent in a new area. In view of the high frequency of possible infusion reactions, serum at baseline and 26 weeks was assessed for the presence of anti-infliximab autoantibodies. Five subjects (33.3%) were positive, with titres ranging between 40 and 2560. All of these showed significant inhibitory activity against infliximab.29 Five (33.3%) subjects were inconclusive for antibody status due to the presence of circulating drug in those samples, which could potentially interfere with detection. The frequency of positive results was much higher (five out of seven; 71%) in patients suffering possible infusion reactions than in those in whom no infusion reaction was suspected (one out of nine; 11%, p = 0.025).

Modified Rodnan skin score

Overall, there was no statistically significant change in MRSS during the study. However, MRSS did fall during the trial with a median value of 22 (range 6–48) at 26 weeks compared with 26 (range 11–45) at baseline assessment. Two other time points were assessed and both provide additional information. At 6 weeks there was deterioration in median MRSS, and this time point had the highest median skin score at 29 (range 11–44). This deterioration suggests that if there is any improvement in MRSS after infliximab it does not occur within the first 6 weeks and also confirms that an active cohort of cases was recruited. Most intriguingly, there was a marked fall in median MRSS at 22 weeks, to 17 (range 6–46). This showed a trend towards significance by non-parametric statistical testing (p = 0.10). Only nine cases (66%) received a full course of treatment and a significant number of subjects developed anti-infliximab antibodies. It is possible that there was a transient benefit from study drug that did not persist at the 26-week time point. The change in the median skin score for the study is summarised in fig 1.

Figure 1

Change in skin score during treatment with infliximab. The median modified Rodnan skin score (MRSS; with interquartile range distribution and range of values at each time point) is shown at baseline, 6 weeks, 22 weeks and 26 weeks. There was a trend towards a decline between the peak skin score at 6 weeks and the 22-week time point, which corresponded to the maximum number of study subjects within 8 weeks of receiving infliximab treatment. Analysis by Wilcoxon signed rank test. Change in median MRSS during study.

Other clinical outcomes

Other clinical variables showed baseline values very typical of a cohort of active dcSSc. There was substantial functional impairment and disease severity as assessed by physician global VAS. For all clinical outcome measures there was overall improvement between baseline and week 26, although not reaching statistical significance. These changes in clinical outcomes during the study are summarised in table 3.

Table 3

Clinical and serological outcome data

Serum biomarkers

The serum biomarkers selected for analysis were chosen to indicate collagen biosynthesis, immunological activity and vascular damage. The level of PIINP was significantly above the normal range, as expected. There was a statistically significant fall in the PIINP level at 26 weeks. There was no change in serum IL-2R levels, a marker of T-cell activation. Interestingly, the absolute values at baseline, in contrast with other reports were not elevated compared with published normal ranges, although a specific control cohort was not included in the present study. Levels of von Willebrand factor (vWF) were reduced at week 26 compared with baseline values, but this change did not quite reach statistical significance (p = 0.056). These data are summarised in table 3.

Skin biopsy analysis

Immunostaining confirmed the variable expression of TGFβ1 in skin biopsy sections. In general, levels of TGFβ1 were higher in uninvolved than in involved skin and more intense epidermal staining was often present at the 26-week assessment. The significance of this is uncertain. Representative skin biopsy sections are shown in fig 2 together with semiquantitative assessment, which is summarised in table 4.

Figure 2

Type I procollagen secretion by explanted fibroblasts at baseline and 26 weeks. The secretion of type I procollagen was assessed by Western blot analysis of fibroblast culture supernatants following concentration by ammonium sulphate precipitation. (A) Individual paired cultures for lesional skin biopsies at baseline and 26 weeks (n  =  12). (B) Representative western blot data are shown together with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control for the cell number. Data summarise three independent experiments. Type I procollagen secretion by explanted fibroblast cultures. Representative data from six paired fibroblast culture supernatant samples at baseline and 26 weeks. RDU, relative densitometry unit.

Table 4

Semiquantitative scoring of TGFβ1 immunostaining skin biopsies

Secretion of type I procollagen by fibroblasts provided an opportunity to assess the potential effect of infliximab administration on this hallmark abnormality in dcSSc skin samples. Paired cultures were available for 10 of 16 subjects—the fibroblast cultures were technically unsatisfactory in the other samples. There was a statistically significant fall in type I procollagen in culture supernatants at 26 weeks. These data are summarised in fig 3.

Figure 3

Immunostaining of involved and uninvolved skin biopsies for transforming growth factor beta 1 (TGFβ1) at baseline and 26 weeks. Representative sections from skin biopsies stained for TGFβ1 by immunochemistry. Positive staining is shown in pink with blue haematoxylin counterstain. Staining was generally weakest in early involved skin. Arrows show sites of strong dermal or epidermal TGFβ1 localisation. The intensity of the staining in the epidermis and dermis was scored by an experienced observer blinded to biopsy details. An IgG incubated control is also shown (inset panel). Staining of systemic sclerosis samples was not consistently more intense than matched healthy control samples (data not shown). Immunostaining for TGFβ1 in skin biopsies.

Discussion

Our trial was essentially a safety study of infliximab in a disease group that had not previously been evaluated. We have explored the safety and tolerability of infliximab therapy in a well-defined cohort of dcSSc cases using a dose of 5 mg/kg with five scheduled administrations over 22 weeks. This treatment regimen is successful in the treatment of Crohn’s disease, a condition in which, like dcSSc, fibrosis is a prominent pathogenic process.

All study subjects were in the progressive phase of SSc evidenced by documented progression of the skin score30 as this was considered to be particularly appropriate for treatment with infliximab. This is a somewhat different disease population compared with cohorts entering other recent studies of dcSSc, in which the duration of disease and threshold skin score has been the major selection criteria. One of the confounding aspects of previous trials has been the tendency for the skin score to improve over time in placebo-treated cohorts. This makes the interpretation of studies very difficult. For example, recent experience in a trial of anti-TGFβ1 monoclonal antibody showed an overall improvement in a cohort of 36 cases of dcSSc, with the extent of improvement correlating inversely with disease duration at study entry.31 In the present trial, there was a trend towards improvement in the median MRSS at the 22-week assessment, compared with the increase in skin score over the first 6 weeks of the study. The high frequency of infusion reactions meant that many subjects received a shorter than planned treatment period. Together, these observations suggest that infliximab may have had a beneficial effect on MRSS in some cases in the early part of the study but this was not sustained at 26 weeks.

Although there were significant AE these were generally attributable to active dcSSc, with only a minority judged to be related to infliximab. The high frequency of anti-infliximab antibody generation, with neutralising activity is somewhat surprising. Previously it has been suggested that these antibodies were formed only at low frequency when infliximab is administered at 5 mg/kg,32 and so the development of these in one third of cases tested is significant and perhaps related to a particular propensity to antibody formation in active dcSSc. In contrast with the findings in other clinical trial populations33 these anti-infliximab antibodies were associated with the occurrence of suspected infusion reactions, necessitating premature withdrawal from the study and also may have attenuated the efficacy of infliximab in this study cohort. This may be relevant to the apparent transient benefit seen with infliximab at 22 weeks. In the future it may be appropriate to co-administer immunosuppressive agents with infliximab if it is to be used in dcSSc, as is current practice in the treatment of rheumatoid arthritis.34

In the present study we observed a significant reduction in type I collagen secretion by lesional fibroblast cultures taken at 26 weeks compared with baseline. However, in the absence of non-treated control cases it is difficult to assess the significance of this finding. As expected, there was substantial variability in change with some cultures showing increased levels of procollagen secretion at 26 weeks. The expression of TGFβ1 in skin biopsies has been studied in a number of previous studies.35 In the present study we examined TGFβ1 protein expression but did not demonstrate any consistent effect of infliximab on dermal or epidermal expression.36

Although there are no validated biomarkers that are of confirmed value in assessing dcSSc cases, the three serological tests selected as outcome measures in this trial are those that were recommended by a consensus panel of experts.24 It is noteworthy that a fall in PIIINP occurred during the study period, suggesting a reduction in new collagen formation.35 No change in serum IL-2R was observed in this study. This suggests that there is not a significant effect of infliximab on T-cell activation in SSc. There was a strong trend for levels of vWF to fall during the study. Elevated levels of vWF have been reported in previous studies of SSc37 and have been associated with increased disease severity.

The main limitations of this study are the absence of a control group and the small sample size. Although a transient fall in skin score with subsequent worsening is consistent with a treatment effect, comparable effects have been reported in other recent studies,38 including open-label studies of immunoablation and autologous stem cell reconstitution.39 In conclusion, we have treated active dcSSc cases with short-term infliximab treatment and observed a trend of an improvement in the skin score and an improvement in two biochemical markers of fibrosis during the study. However, in view of the high frequency of possible infusion reactions we do not recommend further evaluation of the regimen used in this study. However, studies examining other TNFα blocking agents, or the co-administration of infliximab with methotrexate, a recommended treatment for skin disease in dcSSc in the EULAR/EUSTAR guidelines, may be worthwhile.

REFERENCES

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Footnotes

  • Funding This trial was sponsored by Royal Free and University College Medical School. Research in the department is supported by the Arthritis Research Campaign, Raynaud’s and Scleroderma Association and the Scleroderma Society (UK). The authors also obtain support from the Scleroderma Research Foundation (USA).

  • Competing interests Declared. This investigator-initiated study was supported by a research grant from Centocor, Inc, who also provided the study medication.

  • Ethics approval The protocol and patient informed consent forms were approved by the responsible ethics committees at participating sites.

  • Patient consent Obtained.