A Prospective Study Examining the Effect of Selected Topical and Systemic Drugs on Pruritus Grading System Score and STAT 6 Expression in Patients of Prurigo Nodularis.

Background: Prurigo nodularis (PN) is a chronic dermatologic condition presenting as multiple papulonodular lesions occurring with intense pruritus. Though numerous agents (topical, systemic, phototherapy and biological drugs) have been tried, the outcomes are variable.
Objectives: The aim of this study was to assess the role of topical and systemic therapies in primary PN by comparing the Pruritus Grading System (PGS) score at baseline and 1 month post-therapy. Materials and
Methods: Of 86 diagnosed cases of PN, 49 cases of primary PN were clinically graded by Pruritus Grading System Score (PGSS), and assessed histopathologically by IHC staining (STAT-1, 3, and 6). Apart from topical agents, oral nortriptyline (mild grade), methotrexate (moderate grade) and thalidomide (severe grade) were administered, whereas doxepin was administered for itching. The PGSS was assessed after 1 month of therapy.
Results: Among 49 patients of PN, the majority of patients showed a significant decrease in PGSS (P = <0.001) in 1 mont, which correlated with STAT-6 expression. The combination of different topical and oral agents resulted in a statistically significant change in severity, though individual drugs did not achieve statistically significant results.
Conclusion: A combination of selected oral and topical agents can effectively control the severity of PN within one month, and this was found to correlate with STAT 6 expression. Copyright:

Introduction

Prurigo Nodularis (PN), also known as Hyde's prurigo,[1] is an intensely pruritic disease of unknown etiology, which manifests as multiple, firm, hyperkeratotic eroded papules and nodules.[2] PN is a subtype of chronic prurigo (CPG) and is believed to be a reaction pattern consequent to chronic scratching arising out of neuronal sensitization to itch and the resultant itch-scratch-itch cycle, which is the critical event in the pathogenesis.[23]

Although various histochemical changes have been described, including neural dermal hyperplasia, role of neurotrophins (NGF and substance P) and histamine, the focus is now on the various subsets of CD4+ cells, including Th1, Th2, and Th17 cells and the Th2 cytokine (IL-4, IL-13, and IL-31), some of which are targets of new drug therapy.[45]

Though data from clinical trials describe preassigned endpoints in treatment naïve cases, in real-life settings, PN patients often need a combination of topical and oral agents. Numerous topical agents (potent topical corticosteroids, calcineurin inhibitor (pimecrolimus), vitamin D analog, capsaicin),[6] phototherapy,[7] and systemic agents (pregabalin, gabapentin, cyclosporine, methotrexate and thalidomide)[891011121314] have been tried with variable results. The newer drugs administered include aprepitant[15] and serlopitant,[16] neurokinin1 inhibitors, and opioid receptor antagonists like naltrexone,[17] which may not have universal clinical applicability due to their cost and variable efficacy across the severity spectrum of PN.

Our prespecified hypothesis was to assess the role of topical and systemic therapies in PN in relation to STAT expression. All cases of primary PN were evaluated clinically and histopathologically and assessed for severity using the PGSS score.[18] Selected topical and systemic agents were administered, and the improvement after 1 month of therapy was assessed using the Pruritus Grading System Score (PGSS).

Materials and Methods

This prospective study was carried out in the dermatology OPD of a tertiary care hospital from 1st November 2017 to 31st March 2020 after institutional and university ethics approval. All clinically diagnosed cases of PN were assessed to rule out any secondary causes. Immunocompromised patients, pregnant and lactating women, age less than 18 years, and patients on any active treatment for PN were excluded from the study.

Investigations

The tests performed to rule out the secondary causes of PN included a complete blood count, fasting blood sugar levels, liver, and kidney function tests, viral markers (HIV ELISA, hepatitis B, C serology), erythrocyte sedimentation rate, stool for occult blood (to rule out any malignancy), Chest X-ray (to rule out tuberculosis or any other focus of chest infection), and ultrasound abdomen.

Histology and IHC markers

All cases were subjected to a histological assessment using immunohistochemical markers targeted at components of the JAK-STAT cell signaling pathway, which serve as a surrogate marker for T helper cells. IHC markers for STAT-1, 3, and 6 were used to show the activity of Th1, Th17/Th2, and Th2 cells, respectively. (Appendix S1).

Severity grading

PGSS was used to grade the prurigo severity. It has four criteria- distribution, frequency, severity, and sleep disturbance. Based on the PGS score, the disease was graded as mild (score 0 to 5), moderate (score 6 to 11), and severe (score 12 to 19) [Table S1].

Table S1

Pruritus Grading System Score (PGSS)

Distribution	Solitary site	1
	Multiple sites	2
	Generalized	3
Frequency	Episodic	1
	Frequent	3
	Continuous	5
Severity	Rubbing	1
	Scratching	1
	Localized excoriations	3
	Generalized excoriations	5
Sleep disturbance	Rare	0
	Occasional	2
	Frequent	4
	Totally restless	6
Total		19

Therapeutic intervention

All the patients were administered oral antihistamines and super-potent steroids. Based on clinically accepted practice norms in the department, published data, cost considerations, and existing evidenced-based literature, a departmental consensus was arrived to institute systemic agents depending on the severity of PN.[1112131419] The systemic drugs administered were nortriptyline (mild grade), methotrexate (moderate grade) and thalidomide (severe grade), respectively. Doxepin was added if the itching was not adequately controlled by antihistamines. In case there was a lack of response after 2 weeks or in the event of debilitating side effects, the systemic drug could be changed. The PGSS was re-evaluated after 1 month of the therapy.

Statistical analysis

Non-parametric tests were used to make a statistical inference as the data were not normally distributed, and paired Wilcoxon test was used to explore the differences in PGSS at the two time points. Stuart-Maxwell test was used to assess the change in PGS Category between the two time points. A P value of significance was taken as less than 0.05.

Results

Of the 86 clinically and histopathologically proven cases of PN, 49 patients with primary PN were included in the study. The mean PGS score at the time of presentation was 10.71, with the range being 5.0 to 17.0. The patients were followed up, and 1 month later, the mean PGS score was 6.80 (4.0-13.0). The mean fall in PGS score was 3.92 ± 2.06, whereas the mean percent fall in PGS was 35.98 ± 13.23%. [Table 1 and Figure 1] [The demographic profile of patients the histological and IHC findings are detailed in [Table S2].

Table 1

PGSS at baseline and after 1 month of therapy

Category	Mean±SD	P value (Percent Fall in PGSS)
PGSS: Distribution	2.49±0.68	0.196 (N. S.)
PGSS: Frequency	3.10±0.59	0.024
PGSS: Severity	3.16±0.90	0.323 (N. S.)
PGSS: Sleep Disturbance	1.98±1.51	0.987 (N. S.)
Total PGSS at Presentation	10.71±2.68	0.378 (N. S.)
PGSS (1-Month Post-Treatment)	6.80±2.10	<0.001
Fall in PGSS	3.92±2.06	<0.001
Percent Fall in PGSS	35.98±13.23	-

PGSS at 1-Month Post-Treatment and fall in PGSS are statistically significant. NS- Not significant

Figure 1

Fall in PGS score. The Box-and-Whisker plot depicts the distribution of PGS score at presentation with mean score of 10.71 and 1-month post treatment of 6.80

Table S2

Demographic profile of patients the histopathological and IHC findings

Patient characteristics	Number of patients (Percentage)
Age (Mean±SD)	37.49±16.04 years
Gender
Male	33 (67.3%)
Female	16 (32.7%)
Duration of Disease (Mean±SD)	2.69±3.43 years
Lesion Morphology
Excoriated	46 (93.9%)
Intact	3 (6.1%)
Type of lesion
Papule	48 (98.0%)
Nodule	47 (95.9%)
Plaque	21 (42.9%)
Lesion Distribution
Upper Limb	41 (83.7%)
Lower Limb	48 (98.0%)
Trunk	35 (71.4%)
Buttocks	21 (42.9%)
IHC Staining & Histopathology
STAT-1	18 (36.7%)
STAT-3	24 (49%)
STAT-6 positive	35 (71.4%)
S-100 (Case)	6.92±3.40
S 100 (Control)	3.94±2.15
Pseudoepitheliomatous Hyperplasia	10 20.4%
Chronic Inflammatory Infiltrate	100%
Mild	17 (34.7%)
Moderate	28 (57.1%)
Dense	4 (8.2%)
Inflammatory cells	49 (100%)
Lymphocytes	49 (100.0%)
Eosinophils	9 (18.4%)
Plasma Cells	4 (8.2%)
Neutrophils	1 (2.0%)
Vertical Collagen Bundles	25 (51.0%)

The majority of the patients were of moderate severity (PGS score grading 65.3%), followed by severe grade (32.7%) and mild grade (2.0%). [Table 2] When the patients were followed up one month later, 14 (28.6%) patients moved from the moderate to the mild category, 3 (6.1%) patients moved from severe to the mild category, whereas 12 (24.5%) patients moved from severe to moderate category [Table 3]. Thus, majority of the patients remained in the moderate grading (61.2%), whereas the percentage of patients in the mild and severe categories were 36.7% and 2%, respectively [Tables 2 and 3].

Table 2

Distribution of patients in mild moderate and severe groups of PN at baseline and after 1 month of treatment

Category	Mean number of patients (percentage)	P value (Percent Fall in PGS)
PGS Category at Presentation		0.312 (NS)
Mild	1 (2.0%)
Moderate	32 (65.3%)
Severe	16 (32.7%)
PGS Category (1-month post-treatment)		<0.001
Mild	18 (36.7%)
Moderate	30 (61.2%)
Severe	1 (2.0%)

Table 3

Change in PGS Category Over Time (n=49)

PGS Category	Presentation				Stuart-Maxwell test
	Mild	Moderate	Severe	Total	χ 2	P
1-Month Post-Treatment
Mild	1 (2.0%)	14 (28.6%)	3 (6.1%)	18 (36.7%)	26.951	<0.001
Moderate	0 (0.0%)	18 (36.7%)	12 (24.5%)	30 (61.2%)
Severe	0 (0.0%)	0 (0.0%)	1 (2.0%)	1 (2.0%)
Total	1 (2.0%)	32 (65.3%)	16 (32.7%)	49 (100.0%)

The uncoloured cells on the diagonal represent patients whose category did not change. The red shaded cells represent patients who moved to a lower category, whereas the green shaded cells represent patients who moved to a higher category. The IHC marker STAT-6 showed a statistically significant correlation with the percent Fall in PGS (P 0.023%; data not shown)

The overall change in PGS Category was statistically significant (Stuart-Maxwell test: χ2 = 26.951, P = <0.001) [Table 3]. The percentage of fall in PGSS was also found to be statistically significant overall (p-value <0.001%). [Table 1 and Figures 1–4], [Figure S1] The IHC marker STAT-6 showed a statistically significant correlation with the percent fall in PGSS (p-value 0.023).

Figure 4

Change in PGS over time. The bar-graph depicts the proportion of patients in each of the PGS Category at presentation and the change at 1-Month Post-Treatment

(a) Patient with hyperpigmented papulonodular lesions distributed over the upper trunk (severe PGS grade, score 15); (b) Figure shows near clearance of the lesions and improved with a to moderate PGSS grade (score 6) after 1 month of treatment with thalidomide and topical agents

(a) Patient with hyperkeratotic and hyperpigmented papules over both legs (moderate PGSS grade, score 11); (b) Patient improved with a moderate PGSS grade (score 6) after 1 month of treatment with methotrexate

Although we observed that a combination of different topical and oral agents resulted in good treatment response in 1 month, none of the individual treatments revealed a statistically significant association in improving the disease. [Table 4]. Notably, there was a marked improvement in the PGSS after 1 month of therapy [Figure 1], and the change in PGSS correlated with the STAT 6 expression.

Table 4

Results of the individual drugs and correlation with P

Name	Percentage of patients (n=49)	P (fall in PGS)	P (percent fall in PGS)
TOPICAL TREATMENT
Super potent Steroids	100%	-	-
Emollients	53.1%	0.0926	0.984
Camphor + Menthol Lotion	77.6%	0.102	0.175
Salicylic Acid	18.4%	0.915	0.560
Other Topicals
Moisturizing Syndet	6.1%
Capsaicin Cream	4.1%
Calamine Lotion	2.0%
Systemic Treatment
Antihistamines	100%	-	-
Nortriptyline	63.3%	0.645	0.194
Thalidomide	16.3%	0.834	0.329
Doxepin	8.2%	0.206	0.111
Methotrexate	8.2%	0.462	0.770
Dapsone	6.1%	0.651	0.900
Amoxicillin + Clavulanic Acid	20.4%	0.162	0.268

Discussion

It is pertinent to note that although the exact initiating event in PN is not yet certain, the vicious itch-scratch-itch cycle perpetuates the disorder.[123] PN could be primary in origin, without any apparent initiating cause, or secondary to other systemic pruritogenic causes. We had excluded the secondary causes as in these, the efficacy of medical therapy is difficult to ascertain, as it is influenced by the background etiological causes.

Multiple Th2 cytokines such as IL-4 and IL-31 have been implicated in PN, and we used STAT markers to delineate their role.[20] The therapeutic intervention are varied and are based on their modes of action targeting the various pathogenetic aspects of PN. Of the numerous topical agents, [Table 5] the most common drugs used were steroids (100%), followed by camphor/menthol lotion (77.6%) and moisturizers (53.1%).[2122]

Table 5

Topical treatment used in prurigo nodularis and their rationale[2122]

Name	Rationale/Mechanism of action	Dose
Super potent Steroids	Anti-pruritic effect	Once a day application
	Have immunosuppressive effect on T cells
	Modulate release of pro-inflammatory cytokines and neuropeptides such as substance P and CGRP.
Emollients	Reduce xerosis cutis and enhance skin hydration, leading to amelioration of vicious itch- scratch cycle.	2-3 times a day
Camphor + Menthol Lotion	Menthol has a cooling, antipruritic, analgesic and antiseptic action, which is mediated by activation of TRPM8 and TRPA1.	Twice a day
	Camphor is an agonist of TRPV3 and other TRP channels and has cooling and antipruritic action.
Salicylic Acid	Has keratolytic action and helps to reduce hyperkeratosis in PN.	Twice a day
	Salicylic compounds also reduce serotonin induced scratching through the slow release of acetylsalicylic acid.
Moisturizing Syndet	To reduce xerosis and thus induction of pruritus	Once a day
Capsaicin Cream	Nerve sensitization and neural modulation of itch by binding to heat activated ion channel receptor TRPV1 expressed in sensory nerves and keratinocytes.	Twice a day
	Excitation of C nerve fibers causes the release of substance P but prolonged repeat applications deplete substance P and other neurotransmitters. Hence, chronic or high dose use induces nerve ending degeneration and allows the alleviation of itch.
Calamine Lotion	Soothes skin, hence anti- pruritic.	Twice a day

TRP - Transient receptor channels. TRPV1 is transient receptor potential vanilloid 1. CGRP - calcitonin gene-related peptide

Of the various oral treatments, [Table 6] antihistamines were used in all cases, followed by nortriptyline, thalidomide and doxepin. Antiepileptics and antidepressants, including pregabalin and amitriptyline, have been studied in PN.[89192324] Although pregabalin modulates neural gamma-aminobutyric acid (GABA) signaling, antidepressants, including selective serotonin reuptake inhibitors (SSRI) act by a yet unclear mechanism to modulate itch.[6819] Our experience with pregabalin 75 mg daily is not heartening, and hence we did not use this molecule. Zalaudek et al.[23] showed the beneficial effect of amitriptyline with a clinical improvement in 17 PN patients treated with doses ranging from 10 to 60 mg for 6 weeks. We used nortriptyline 25 mg, and found this drug tolerable and an effective adjuvant. We found thalidomide to be effective in refractory PN at a low dose of 50-100 mg/day with minimal side effects, making it a clinically useful drug.[1314] Methotrexate has been used by multiple authors in a dose varying from 5-25 mg with a disease response of 91-94% at 3-6 months.[1112] Doxepin was used for its profound antihistaminic action.

Table 6

Systemic treatment used in prurigo nodularis and their rationale[689192324]

Drug	Rationale/mechanism of action	Dose
Antihistamines	There are increased mast cells in PN lesions which can release histamine and other pruritic mediators like tryptase and prostaglandins	Ebastine 10-20 mg/day
Nortriptyline	Tricyclic antidepressant	Nortriptyline 25-50 mg HS
	Antipruritic effect based on a high binding affinity for the histamine H1 receptors and due to serotonin reuptake blockade of the peripheral nociceptive C-fibres.
	Sedative effect, which decreases the restless activity of continuous scratching of the skin.
Thalidomide	Immunomodulator, central depressant, anti-inflammatory and neuromodulatory action.	Thalidomide 50-100 mg per day
	Inhibits TNFα production and NF-?β cascade, upregulates TGF-β production and keratinocyte proliferation and migration, and stimulates re-epithelization, which contributes to wound healing.
	It may affect type C unmyelinated fibers involved in neural pathways
Doxepin	Tricyclic antidepressant.	Doxepin 25 mg OD (or BD)
	Antagonist for H1 and H2 receptors, 5HT1 and 5HT2 serotonergic receptors, α1 adrenergic and muscarinic receptors.
	Functions as antipruritic, anxiolytic, sedative, hypnotic and antidepressant agent
Methotrexate	Downregulation of IL-6, IL-12, TNF and IFN-gamma	7.5-15 mg once/week
	Impairment of neutrophilic chemotaxis,
	Selective apoptosis of proliferating CD4+ cells
	Inhibition of IL-1 activity and neovascularization
	Immunosuppressive effect on epidermal LCs (shown in vitro)
	Through its direct effect on lymphocytes, epidermal Langerhans cells and cell adenosine metabolism, MTX can selectively act on the main pathways of PN.
Dapsone	Anti-inflammatory and antibacterial agent	100 mg/day
Amoxicillin + Clavulanic Acid	To control secondary infection	625 mg TDS X 5 days

TNF - tumor necrosis factor

At doses used, most patients responded to the treatment with minimal side effects. The most commonly observed adverse effect was sedation, which was seen with thalidomide (5 of 8 patients), nortriptyline (12 of 31 patients), and doxepin (2 of 4 patients). Constipation was seen in one patient with the use of thalidomide. Steroid induced atrophy was seen in 2 patients in the perilesional skin, attributable to the inappropriate method of application. No side effects were reported with other drugs in our cohort of patients.

We noted that no single drug achieved a significant decrease in PGS score by 1 month, [Table 4] which suggests that expectations of any single drug being effective in PN is an unrealistic proposition in real-world clinical practice, and combination therapy is more useful in PN.

A notable aspect is the increased expression of STAT 6 in lesional PN skin, which is a marker for Th2 cells[20] which releases IL-4,10,13. These cytokines are the focus of novel therapies in PN like Dupilumab, a monoclonal antibody that targets the α chain of the IL-4 receptor, and Nemolizumab, which targets the IL-31 receptor. However, the use of these biologicals is limited by the high cost and relative unavailability of the drug in all countries. Hence, in a resource-limited setting, a combination of topical and systemic agents may achieve comparable results as the expensive biological drugs.

Recently, it has been observed in PN that the IL-22 levels were upregulated, and the source of these IL-22 were CD4+ and CD8+ cells. RNA sequencing was done by Belzberg et al.[25] to reveal markedly upregulated Th22 genes in lesional skin, as compared with non-lesional skin. Also, the expression of IL-22 receptors- IL22R1 and IL22R2 were also upregulated in lesional PN skin. Thus, Th22 cells are also likely to have a pathogenic role in PN, which is mediated by STAT3 receptors. However, we did not find STAT3 to correlate with the severity.

There are drawbacks in our study as we did not assess the clinical and IHC parameters after 1 month of therapy which is relevant with drugs like methotrexate, where a longer duration would lead to a favorable response. But in real-life scenarios, a patient is in need of emergent treatment, and outside the strict rubric of a trial, we do not feel that a delayed onset of results is desirable or practical. Although side effects are a concern and may appear with prolonged therapies, most of the drugs we used did not show any major side effects. We reiterate the existential fact that there are multiple mechanisms that regulate and perpetuate PN, and thus, it is imperative to use multiple drugs, and a single drug may be inadequate for treatment response, which was elegantly shown in our study. Also, it would be ideal to assess the action of individual drugs on STAT expression as that would help to understand which agent specifically acts via these receptors and can help re-purpose existing drugs for PN.

Our study reiterates that the Th2 pathway is predominantly involved in PN, making the use of biological drugs like Dupilumab an attractive option. However, in resource-poor settings, the use of such expensive drugs becomes incapacitating. Our study points out that re-purposing conventional drugs, like nortriptyline, methotrexate, and thalidomide, work remarkably well in reducing the disease burden and correlate with the immunological expression of Th2 cells/STAT 6.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

(a) Patient of prurigo nodularis with multiple papulonodular lesions distributed over bilateral lower limbs (moderate PGS grade, score 8); (b) Figure showing the improvement in the clinical lesions (mild PGS grade, score 3) after 1 month of treatment with nortriptyline and topical agents

Supplementary File

Grading

Mild grade- If total score is between 0 and 5

Moderate grade- If total score is between 6 and 11

Severe grade- If total score is between 12 and 19