Abstract
Background:
Oral pemphigus vulgaris (PV) presents with persistent, painful, nonhealing oral erosions, with a slower treatment response compared to cutaneous lesions.
Patients and Methods:
To assess the efficacy of adjuvant topical rituximab encapsulated in nanoparticle gel in oral PV. Of 31 oral PV patients recruited, 16 were randomized to rituximab incorporated in calcium alginate nanoparticles (study group) and 15 to calcium alginate nanoparticle gel only (placebo group), applied twice daily on oral erosions for 12 weeks. Both groups, in addition, received oral prednisolone (tapering doses) and azathioprine.
Results:
In the topical rituximab group, 75% of patients and in the placebo group, 86.7% of patients achieved remission (P = 0.65) over 12 weeks of therapy. The median time to remission in the topical rituximab group was 4 weeks, and in the placebo group, it was 8 weeks (P = 0.53). Median oral pemphigus scores showed an earlier reduction in the rituximab group (P = 0.52). The median circulating CD20 count was reduced in the topical rituximab group from 100 to 74/mm3 at week 8. In the placebo group, the value increased from 89 to 118/mm3 (intergroup P = 0.29). A significantly greater number of cases in the topical rituximab group had a negative mucosal CD20 count (P = 0.001). There was a comparable fall in desmoglein 3 titers in both groups (P = 0.58). The mean cumulative prednisolone dose was 3567 mg in the topical rituximab group and 3869 mg in the placebo group (P = 0.125). No side effects of rituximab were observed.
Limitations:
A small sample size, and need of pharmacokinetic studies in animal models to assess absorption of rituximab in nanogel formulation.
Conclusion:
With topical rituximab incorporated in nanogel, there was a trend toward earlier remission, lower total cumulative oral prednisolone dose, earlier improvement in oral pemphigus score, and greater reduction in CD20 count.
Keywords: Nanogel, oral pemphigus vulgaris, topical rituximab
Introduction
Oral erosions of pemphigus vulgaris (PV) cause significant morbidity. Rituximab infusion is now recommended as first-line therapy in PV.[1,2] Intralesional rituximab has been tried in a small case series in refractory oral PV with encouraging results.[3] We undertook this pilot study to assess the utility of the topical application of rituximab, encapsulated in a nanoparticle gel formulation as an adjuvant in oral PV.
Patients and Methods
This pilot study was undertaken as a single-center, prospective, randomized, double-blind, placebo-controlled trial on 31 oral PV cases, post institutional ethics committee approval (IEC 47/29.11.2017). Consenting patients 18–72 years of age, with predominant oral PV (with or without cutaneous involvement of body surface area ≤1%), confirmed by enzyme-linked immunosorbent assay (ELISA) test for desmoglein (dsg) 3 antibody and mucosal biopsy, were included. The severity of oral PV using objective and subjective assessment parameters, including modified Saraswat score (MSS), investigator global assessment (IGA; photographic documentation of percentage change in oral erosions, with grading from −1 to 5), and patient global assessment (PGA; by visual analog scale from 0 to 100, 0 meaning no improvement in disease and 100 meaning complete improvement) were done at baseline and each follow-up visit. Photographic documentation of mucosal lesions was done using a mobile camera (OnePlus 6 model, primary camera: 16 megapixel, 1.7 sensor) with fixed background and lighting conditions. Specific investigations included a mucosal biopsy for histology and CD20+ B cell count (by immunohistochemistry [IHC]), as well as an ELISA test for serum anti-dsg3 antibody titer at baseline and week 8. IHC reading was undertaken by a pathologist who was blinded to pre-and post-treatment biopsies. The intensity of the infiltrate was graded as follows:
1+: Mild perivascular infiltrate with/without interstitial infiltrate
3+: Dense perivascular infiltrate with/without interstitial infiltrate
2+: Density of inflammatory infiltrate between 1+ and 3+
Patients were randomly allocated to the study or placebo group. Randomization was done by generating two sets of random numbers using the website www.randomizer.org. Allocation concealment was done using the envelop method. The patients were followed up biweekly in the first month, then monthly for the next three months, and subsequently every three months for one year.
Study group patients applied topical rituximab incorporated in a calcium alginate nanoparticle gel, and the placebo group applied calcium alginate nanoparticle gel only twice a day for 12 weeks or until oral ulcers healed, whichever was earlier. Additionally, both groups received standard treatment with a tapering schedule of oral prednisolone (initiated at 1 mg/kg/day up to a maximum of 60 mg/day) and azathioprine sodium, 50 mg twice daily.
Rituximab-loaded nanoparticles were prepared at the nanoparticle laboratory of our institute. Alginate nanoparticles were obtained by inducing gelification of sodium alginate (Sigma-Aldrich, W201502) using calcium chloride (Merck, 1.02378.0500) followed by stabilizing with poly-l-lysine (Sigma-Aldrich, P8920) which forms a poly-electrolyte complex. Drop-wise addition of commercially available rituximab for intravenous infusion (10mg/ml) formed stable “rituximab loaded alginate nanoparticles.” This preparation was stored at 4–8°C in packed containers. The nanoparticles were characterized for size using transmission electron microscopy and Nanosight. The average size of nanoparticles was 200 ± 15 nm. Bradford assay of the supernatant was done to measure the rituximab concentration of the alginate nanoparticles, which showed a 67.72% loading of rituximab within the nanoparticles. The final concentration of rituximab achieved was 1 mg/ml. The study drug and placebo were packed in airtight, sealed 30 ml plastic bottles. To maintain drug stability, these bottles were carried by patients in ice packs and kept in the refrigerator at 4–8°C. Patients were instructed to apply 15 drops (amounting to 1 ml) of drug or placebo twice daily, and adherence to treatment was periodically checked.
Statistical analysis
It was done using Stata 16.0 (StataCorp LP, Texas, USA). Data was presented as number (percentage), mean (±standard deviation), or median (range: minimum–maximum) as appropriate. Baseline characteristics were compared using Fisher’s exact test/Wilcoxon rank-sum test. The intention-to-treat analysis was performed for primary outcome measures, and per-protocol analysis was done for secondary outcome measures. The primary outcome was compared between the groups using the Kaplan–Meier curve followed by the log-rank test. The hazard ratio and 95% confidence interval (CI) were also calculated to assess the effect of the intervention. The secondary outcomes were compared between the groups using the Wilcoxon rank-sum test (for variables that did not follow a normal distribution) and Fisher’s exact test (for categorical variables). A P value < 0.05 was considered significant.
Results
Of the 86 patients screened, 31 cases fulfilling the inclusion criteria were included. Of these, 16 cases were randomly assigned to the study group and 15 cases to the placebo group. Baseline characteristics were comparable [Table 1]. Two patients in the rituximab group and one in the placebo group were excluded due to disease flare and development of azathioprine-induced leucopenia, and uncontrolled hyperglycemia, respectively. In the rituximab group, 12 of 16 (75%) patients, and in the placebo group, 13 of 15 (86.7%) patients achieved remission over 12 weeks of therapy (P = 0.65). The Kaplan–Meier curve showed the median time to remission in the study group to be 4 weeks (95% CI: 2–8 weeks) and in the placebo group, it was 8 weeks (95% CI: 4–8 weeks) (log-rank P = 0.53) [Figure 1]. The reduction in median modified Saraswat score (MSS) for oral functional disability at each follow-up from baseline was significant (P < 0.05) in both groups. There was an earlier reduction in investigator and patient global assessment scores in the study group compared to the placebo, but they were comparable at week 12 [Figure 2a and 2b]. The median circulating CD20 count fell from 100/mm3 at baseline to 74/mm3 at week 8 in the rituximab group but increased from 89 to 118/mm3 (intergroup P = 0.29) in the placebo group. We found a significant decline in tissue CD20+ lymphocytes with both topical rituximab (P = 0.004) and placebo (P = 0.04). However, 11 (78.5%) cases became negative for CD20+ lymphocytes at week 8 in the rituximab group, while only 2 (14%) became negative in the placebo group (P = 0.001). There was a significant and comparable reduction in median ELISA titers in both groups at week 8. The mean total cumulative dose (TCD) of oral prednisolone in the study group at week 12 was 3567 mg (±833), and in the placebo group, it was 3869 mg (±699) (P = 0.12) [Table 2].
Table 1.
Baseline characteristics of patients in the study
| Parameters | Topical rituximab group (n=16) | Placebo group (n=15) | P |
|---|---|---|---|
| Pure mucosal involvement | 12 (75%) | 13 (86.6%) | 0.65 |
| Cutaneous involvement less than 1% body surface area | 4 (25%) | 2 (13.3%) | 0.65 |
| Other mucosal involvement | 2 (12.5%) | 0 | 0.48 |
| Weight (kg) before the study [mean (±standard deviation)] | 57.9 (±15.4) | 60.6 (±11.2) | 0.58 |
| Baseline oral pemphigus score: median (range) | 54.75 (27–60) | 54 (19.5–60) | 0.63 |
| Objective (0–17) | 13 (3–15) | 11 (4–15) | 0.39 |
| Subjective (0–45) | 43.5 (21–45) | 42 (8.5–45) | 0.82 |
| Number of patients whose Tzanck smear showed acantholytic cells n (%) | 15 (93.7%) | 15 (100%) | 1.00 |
| Number of patients whose mucosal biopsy consistent with pemphigus vulgaris n (%) | 8 (50%) | 7 (46.6%) | 1.00 |
| Baseline anti- dsg3 ELISA (U/ml), median (range) | 353 (7–441) | 263 (13.8-438) | 0.43 |
| Baseline circulating CD20 count (per cubic mm), median (range) | 100 (26–350) | 89 (22-564) | 0.87 |
| CD20 count in oral mucosal biopsy by IHC | 1.00 | ||
| Grade: n(%) | 3+: 7 (50%) | 3+: 7 (50%) | |
| 2+: 6 (43%) | 2+: 7 (50%) | ||
| 1+: 1 (7%) | 1+: 0 (0%) |
ELISA: Enzyme-linked immunosorbent assay, IHC: Immunohistochemistry
Figure 1.
Kaplan–Meier curve showing the median time to achieve remission in the two study groups
Figure 2.
(a) Topical rituximab group—Baseline—Total score 54.5 (objective: 10, subjective: 44.5), IGA: 0, PGA: 0; (b) At 12 weeks: Total score 2 (objective: 2, subjective: 0), IGA: 5, PGA: 90
Table 2.
Results in the two treatment groups
| Topical rituximab group n=14 | Placebo group n=14 | P | |
|---|---|---|---|
| Oral pemphigus score (0–62) [(median (range)] | |||
| Baseline | 54.75 (27–60) | 54 (19.5–60.0) | 0.48 |
| Week 2 | 8.5 (0–53) | 20 (0–53) | 0.15 |
| Week 4 | 3.5 (0–47) | 6 (0–46) | 0.21 |
| Week 8 | 3 (0–20) | 3 (0–13.5) | 0.51 |
| Week 12 | 0 (0–19) | 0 (0–10.5) | 0.52 |
| Investigator global assessment (-1-5) [Mean± standard deviation] | |||
| Baseline | 0 | 0 | 0.21 |
| Week 2 | 3.64±1.28 | 3.1±1.13 | 0.91 |
| Week 4 | 4.0±0.99 | 4.1±0.92 | 0.85 |
| Week 8 | 4.5±0.94 | 4.5±0.65 | 0.63 |
| Week 12 | 4.7±0.43 | 4.8±0.36 | |
| Patient global assessment (0-100) [Median (range)] | |||
| Baseline | 0 | 0 | 1.3 |
| Week 2 | 65 (10–100) | 50 (0–90) | 0.77 |
| Week 4 | 80 (15–100) | 70 (40–100) | 0.44 |
| Week 8 | 90 (45–100) | 80 (50–100) | 0.87 |
| Week 12 | 100 (50–100) | 100 (70–100) | |
| CD20 count in blood (per cmm) [Median (range)] | |||
| Baseline | 100 (26–350) | 89 (22–564) | 0.87 |
| Week 8 | 74 (11–535) | 118 (21–571) | 0.29 |
| P | 0.97 | 0.63 | |
| CD20+ lymphocytes in mucosal tissue by immunohistochemistry | |||
| Baseline | 7 | 7 | 1.0 |
| 3+ | 6 | 7 | 0.001 |
| 2+ | 1 | 0 | |
| 1+ | |||
| Week 8 | 0 | 0 | |
| 3+ | 0 | 8 | |
| 2+ | 3 | 4 | |
| 1+ | 11 | 2 | |
| Negative | 0.004 | 0.044 | |
| P | |||
| Desmoglein 3 antibody titers (U/ml) [Median (range)] | |||
| Baseline | 353 (7–441) | 263 (13.8–438) | 0.43 |
| Week 8 | 187.5 (0–423) | 133 (8–477) | 0.58 |
| P | 0.015 | 0.013 | |
| Total cumulative dose of oral corticosteroid (mg) | 3567 (±833) | 3869 (± 699) | 0.12 |
| Mean (±standard deviation) |
Discussion
Given the significant morbidity in mucosal-dominant PV patients and the slow response observed with various medications, especially topical agents, there is a pressing need for alternative agents. Various local therapies, including topical and intralesional corticosteroids, topical cyclosporine, tacrolimus, sirolimus, pilocarpine, and intralesional platelet-rich plasma (PRP), have shown moderate response.[4,5,6] Intralesional triamcinolone acetonide (ITA) used as an adjuvant to oral corticosteroid in oro-pharyngeal PV showed earlier remission (126.6 days with ITA versus 153.2 days without it) and a smaller TCD of oral corticosteroid (4894 mg versus 5312 mg), but caused pain and mucosal atrophy.[7] In a retrospective cohort study of five mucosal PV cases who were initially given only topical agents (clobetasol 0.05% gel, clobetasol 0.05% rinse, tacrolimus 0.1% ointment), four patients achieved disease control after mean follow-up of 8 weeks, but exacerbated later and eventually all patients had to be given systemic immunosuppressants.[8] In another study of 35 cases of oropharyngeal PV, 16 patients who received oral corticosteroid, adjuvant (azathioprine/cyclophosphamide), and intralesional triamcinolone achieved faster remission than those on oral therapy alone (126 days [±41] versus 153 days [±97]).[7]
Intravenous rituximab has replaced other agents in the treatment of PV over the last decade. Intralesional rituximab was used in three oral PV cases, where two doses of 5 mg/cm2 were given on day 1 and day 15. A response was observed in all cases, with a marked decline in circulating CD19 cell count on day 15.[3] But this is an invasive procedure, and similar to rituximab infusion, these patients required premedication in the hospital. Hence, the research question in our study was whether this molecule, dispensed as a topical nanogel formulation, would be beneficial in mucosal erosions of PV. The alginate nanoparticle has good mucoadhesive properties and has been found effective in delivering drugs in mucosal tissues, including oral ulcers.[9] Each milliliter of rituximab-loaded hydrogel contained 1 mg of rituximab. The TCD of rituximab per patient was 60 mg per month. In the study on intralesional rituximab, the cumulative dose of rituximab injected was 60–100 mg.[3] Hence, the cumulative dose delivered by the topical rituximab formulation over a period of 1 month in our study seems adequate. The topical rituximab formulation had many advantages. It was easy to apply as home-based therapy, did not require any premedication, was noninvasive, and was painless. No adverse reactions were reported with our preparation. In both groups, standard systemic treatment for oral PV was co-prescribed for ethical considerations, as this is a pilot study, and response to topical rituximab used alone is unknown. Our study showed that with topical rituximab incorporated in nanogel, there was a trend toward earlier remission, a faster reduction in functional disability scores, a lower TCD of prednisolone, and a greater reduction in CD20 count.
Limitation
A limitation of the study is that a fixed number of cases were recruited since this was a pilot study, so it may be underpowered to detect a true difference between the two groups. Hence, larger studies conducted over longer follow-up periods are required. Pharmacokinetic studies, including animal model studies on the mucosal absorption of rituximab incorporated in nanogels, are also required.
Conclusion
The addition of topical rituximab in nanogel formulation to conventional treatment provided additional benefit in healing of oral PV erosions with respect to certain efficacy parameters.
Conflicts of interest
There are no conflicts of interest.
Use of artificial intelligence (AI)
The authors confirm that there was no use of artificial intelligence (AI).
Acknowledgments
The authors would like to thank Ms. Preeti Sharma, Department of Dermatology and Venereology, AIIMS, New Delhi, for performing anti-dsg3 ELISA and Ms. Aksha and Ms. Shiwangi, Department of Nanotechnology, AIIMS, New Delhi, for preparing the drug formulation.
Funding Statement
Nil.
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