Efficacy of Targeted Mast cell Inhibition Therapy in Chronic Prostatitis/Chronic Pelvic Pain Syndrome

Abstract

Objective:

To identify a subgroup of patients with mast cell dysfunction in chronic prostatitis/chronic pelvic pain syndrome and evaluate efficacy of mast cell directed therapy.

Materials and Methods:

Men with CP/CPPS were recruited and evaluated in an open label, interventional uncontrolled trial after therapy with cromolyn sodium and cetirizine hydrochloride. The primary endpoint was a change in mast cell tryptase concentrations after treatment while secondary endpoints were changes in the NIH-CPSI and AUA-SI. Isolated cells from post-prostatic massage urine were evaluated for immune changes using mRNA expression analysis.

Results:

31 patients with a diagnoses of Category III CP/CPPS were consented, 25 patients qualified and 20 completed the study after meeting a prespecified threshold for active tryptase in expressed prostatic secretions. After treatment with cromolyn sodium and cetirizine dihydrochloride for 3-weeks, active tryptase concentrations were significantly reduced from 49.03±14.05 ug/ml to 25.49±5.48 ug/ml (p<0.05). The NIH-CPSI total score was reduced with a mean difference of 5.2±1 along with reduction in the pain, urinary and quality of life sub scores (p<0.001). A reduction in the AUA-SI was observed following treatment (p<0.05). NanoString mRNA analysis of isolated cells revealed downregulation of immune related pathways including Th1 and Th17 T cell differentiation and TLR signaling. Marked reduction in CD45+ cells and specifically macrophages and neutrophil abundance was observed.

Conclusion:

Identification of CP/CPPS patients with mast cell dysfunction may be achieved using tryptase as a discriminating biomarker. Mast cell directed therapy in this targeted subgroup may be effective in reducing symptoms and modulating the immune inflammatory environment.

INTRODUCTION

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a component of urologic chronic pelvic pain syndrome (UCPPS) and affects men, leading to chronic pain in the pelvic region or genitalia that is often accompanied by increased urinary frequency and urgency as well as sexual dysfunction. CPPS has an estimated prevalence rate of 6-12% of the male population with most diagnoses between the ages of 35-45 ^{1, 2}. We have previously demonstrated that mast cells are critical mediators of both chronic pelvic pain and lower urinary tract symptoms (LUTS) in CP/CPPS ^3-5. Mast cell tryptase was observed to be significantly elevated in patients with CP/CPPS compared to controls and was implicated in our animal studies as a potential mediator of pathogenesis ⁶. In the experimental autoimmune prostatitis (EAP) model of CP/CPPS, we demonstrated that functional inhibition of mast cell activity can significantly attenuate pelvic pain as well as symptoms of voiding dysfunction ^{3, 4}. In this pilot clinical study, we sought to identify subtypes of CP/CPPS characterized by mast cell dysfunction and evaluate the therapeutic efficacy of functional mast cell targeting. These pilot studies serve as a necessary precursor to future clinical trials focused on discrete CP/CPPS subtypes that utilize mechanistic therapies and disease associated surrogate biomarkers.

MATERIAL AND METHODS

Study design

The clinical trial utilized single subgroup assignment, which was then evaluated in an open label, interventional uncontrolled study where comparison of mast cell tryptase levels and changes in symptom scores were evaluated before and after therapeutic intervention with cromolyn sodium (200mg qid) and cetirizine hydrochloride (10mg qd). The therapies were used under an exemption from IND regulations by the FDA. Study data has been reported by adhering to the Consolidated Standards of Reporting Trials (CONSORT) extension to pilot and feasibility trials ⁷.

Study population

The clinical study population consisted of males who were at least 18 years old, had previously been diagnosed with Category III CP/CPPS and reported pain and discomfort in the pelvic region for at least a 3-month period within the 6 months immediately before the first baseline screening visit. All patients had a total score of at least 12 of 43 on the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI) at screening. Patients were recruited through Northwestern University Feinberg School of Medicine’s Department of Urology through a combination of referring physicians, advertisements, and patient outreach from Clinicaltrials.gov. Recruited patients were then screened for mast cell tryptase levels in their expressed prostatic secretions. (EPS). The lower threshold value of 25 ng/ml for mast cell tryptase was based on previous preliminary studies from healthy controls where mast cell tryptase values were either below the threshold of detection or at levels that did not exceed 25ng/ml. Men who met the inclusion and exclusion criteria (Supplementary Table 1) were enrolled into the study.

Data and specimen collection

Appropriate NIH-CPSI and AUA-SI questionnaire data, Voided Bladder 3 (VB3), and expressed prostatic secretions (EPS) samples were collected at the department of urology outpatient clinic at Northwestern Memorial Hospital, Chicago IL (NCT01676857). The written consent procedure was reviewed by the Northwestern University IRB Panel D and approved with the IRB number STU00030121. All participants provided written informed consent to participate in this study. VB3 samples and EPS samples were either used immediately or stored at −80°C for future analyses. VB3 samples were centrifuged at 400g for 5 min at 4°C and supernatant removed and stored at −80°C. The pellets were then processed for RNA extraction and NanoString analysis.

NIH-CPSI symptom score and AUA-symptom index

The NIH-CPSI contains 13 items focusing on the 3 domains of pain or discomfort (8 items), urinary symptoms (2 items) and quality of life impact (3 items). An overall NIH-CPSI score of 0 to 43 was calculated per participant by summing the pain (range 0 to 21), urinary (range 0 to 10) and quality-of-life impact (range 0 to 12) sub-scores. A 6-point decrease in NIHCPSI score has been shown to be clinically perceptible in previous clinical trials of men with CP/CPPS ⁸. AUA symptom index includes 7 questions covering frequency, nocturia, weak urinary stream, hesitancy, intermittence, incomplete emptying, and urgency ⁹. Using the AUASI patients with symptom scores of 0–7 were considered to have mild symptoms while 8-19 had moderate and ≥20 had severe symptoms ¹⁰. A 3-point reduction in the AUA-SI is often considered clinically relevant.

Mast cell tryptase quantitation

Mast cell tryptase levels were measured in EPS from CPPS patients using the mast cell degranulation kit (Millipore) following manufacturer’s protocol. EPS samples (5-10 microliter) were incubated with a chromogenic tryptase substrate, compared to recombinant human tryptase standards and results were expressed in terms of relative tryptase levels after adjusting for dilution of samples.

NanoString analysis

RNA was extracted from the VB3 cell pellets from 5 patients pre- and post-treatment using the RNeasy Kit (Qiagen) and concentrated using RNA Clean & Concentrator (Zymo). Low RNA input amplification kit (NanoString) was done using Human Immunology Panel specific primers and RNA from 5 CP/CPPS subjects were used as input for NanoString nCounter hybridization, and expression analysis with the NanoString human immunology panel. Analysis was done with the nSolver Advanced Analysis Module normalized with default settings ¹¹. Differential gene expression analysis was performed with nSolver software using “treatment” as the covariate and utilizing theThe Benjamini-Yekutieli method.

Statistical analyses

Statistical analyses were performed using GraphPad Prism^™ (GraphPad Software, San Diego, CA, USA). Mast cell tryptase analysis was performed using a combination of linear regression and correlation (Pearson) between tryptase values and patient-reported pain, urinary, QOL and total scores. Ratio paired t tests were used when comparing two groups and a mixed model was used for more than two groups instead of a repeated measures ANOVA because of the presence of missing data values. We analyzed the data instead by fitting a mixed model as implemented in GraphPad Prism 8.0. This mixed model used a compound symmetry covariance matrix and was fit using Restricted Maximum Likelihood (REML) with Geisser-Greenhouse correction. In the presence of missing values (missing completely at random), the results can be interpreted like repeated measures ANOVA. Total differences were determined between patients before and after treatment using unpaired T-test. Data are represented as the mean ± standard error of the mean (S.E.M.). # p < 0.1, * p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001.

RESULTS

Participants:

A total of 31 patients who met the NIH criterion for a diagnoses of Category III CP/CPPS were sequentially consented for the study (Supplementary Table 1). Following sample collection, the levels of active mast cell tryptase were quantified in EPS of the enrolled patients. Based on a pre-specified lower threshold of 25ng/ml, a total of 25 subjects (80.6%) were deemed to have qualified for the intervention phase of the pilot trial with 6 subjects (19.3%) designated as screen failures. Of these, 20 (80%) completed the intervention phase of the study and provided questionnaires which were utilized for evaluation of the objectives of the trial (Fig. 1A). Of the 20 subjects, 19 subjects provided an EPS sample before and after treatment while 1 subject was not able to provide a post-treatment EPS sample and was therefore excluded from the analysis of treatment efficacy on tryptase concentrations. 5 subjects (20%) who were designated as withdrawals/failed adherence, did not complete the study due to incorrect medication dosing (3) and scheduling difficulties for the second visit (1). 1 subject (4%) discontinued medication due to an allergic reaction that was reviewed by a study physician and the Northwestern University IRB and deemed to not rise to the level of a serious adverse event. The remaining 20 subjects who qualified for the trial had a median age of 36.7 (43.12±14.81). At the baseline screening visit, these subjects were administered the NIH-CPSI, and AUA-SI and responses were quantified. Qualified subjects reported a median NIH-CPSI total score of 26 (25.23±6.66) with pain sub-score of 11.5 (12.25± 3.84), urinary sub-score of 3.5 (4.0±2.68) and QOL sub-score of 9.5 (8.98±2.47). The median AUA-SI was 11.5 (13.5±8.24). The median active tryptase level at screening in qualified subjects was determined to be 30.33 μg/ml (46.83 ± 60.56). All 20 subjects who completed the study were contacted a week after their final visit and did not report any significant issues following their participation.

Figure 1. Active tryptase concentrations in CP/CPPS patients are reduced following treatment.

A) Distribution of active tryptase concentrations in all consented patients B) Estimation plot of active tryptase concentrations in patients who completed the therapy trial and provided matched EPS samples (n=19) at the start (pre-) and end (post) of the mast cell directed therapy. The two groups were analyzed using ratio paired two-tailed t test and were significantly different (p=0.0095). The mean of differences ± SEM between the two groups (post-pre) is shown on the right y-axis.

Combination therapy reduces mast cell tryptase levels.

We examined the efficacy of a 3-week therapeutic intervention with cromolyn sodium at 200mg qid and cetirizine dihydrochloride at 10mg qd in selected patients with an elevated level of tryptase in EPS. Patients were requested to self-administer the medications at home daily for 3-weeks followed by a return visit where biological sample collection was repeated. Patients responded to the NIH-CPSI and AUA-SI questionnaires at baseline, weekly, and at the end of the treatment period before sample collection. Active tryptase concentration in EPS was measured and showed a statistically significant reduction (p<0.0001) between pre- and post-treatment. Of the 19 patients with complete data sets, 15 (78.9%) responded to the therapy with a reduction in EPS tryptase concentrations. 4 patients (21.05%) did not show a reduction or showed an increase from their baseline tryptase measurement. Active tryptase concentrations were reduced between individual patients at baseline (49.03±14.05 ug/ml) and post-treatment (25.49±5.48 ug/ml). Treatment responders (n=15) showed an average percentage reduction in tryptase concentrations of 63.95±27.97. These results demonstrate that the therapeutic intervention was successful in meeting the primary endpoint of a statistically significant reduction in tryptase concentrations in EPS.

Mast cell direct therapy modulates the prostate immune environment.

We sought to understand the impact of mast cell directed therapy on the immune environment within the prostate following treatment. To do so, we isolated cells from the post-prostatic massage urine (VB3 fraction) of patients before and after treatment and performed RNA extraction to do a NanoString mRNA analysis for immune associated genes. In 15 out of 20 patients, sample analysis was not possible due to insufficient RNA extraction and/or quality control failures during the NanoString analysis and/or absence of matched pre- and post-treatment samples from the same patient. Complete data sets from 5 patients were analyzed and changes in differentially expressed genes were evaluated. All 5 patients were responders to the mast cell therapy as determined by a reduction in tryptase from pre-treatment levels. In comparing pre-treatment to post-treatment, all significantly different mRNA were observed to be down regulated (17 genes) with no mRNA showing significant elevation following treatment (Fig. 2A). CX3CL1, ICAM4, PIGR showed a log two-fold or greater reduction in mRNA expression with several other genes including LLRB3, IDO1, MAPKAPK2, TLR8, CXCR1, KIR3DL3, CCBP2, TYK2, FN1, TNFRSF14, FCGRT also showing significant (p<0.05 or p<0.1) reductions (Fig. 2B). ATG5, an autophagy related gene was the only gene that was found to be significantly upregulated (p<0.1) following treatment. We next analyzed gene expression signatures of the pre- and post-treatment samples on known immune-related pathways. With exceptions such as chemokine and cytokine signaling, treatment resulted in a down-regulation of the majority of immune related pathways including Th1 and Th17 T cell differentiation and TLR signaling which have been previously implicated in pathogenesis (Fig. 2C). We also evaluated the abundance of different immune cell subsets based on their gene expression signatures in pre- versus post-treatment samples. While mast cell directed therapy appeared to have minimal impact on abundance of dendritic cells, exhausted CD8 T cells or cytotoxic cells, there was a marked impact on CD45+ cells overall and specifically on innate immune cells such as macrophages and neutrophils (Fig. 2D). These data show that mast cell directed therapy in CP/CPPS can significantly reduce both abundance of inflammatory cell populations as well as expression of pro inflammatory gene signatures.

Figure 2. Mast cell directed therapy inhibits immune pathways in CP/CPPS.

A) mRNA expression of immune related genes in cells from the post-prostatic massage urine (VB3 fraction) of 5 patients before and after treatment were analyzed using a NanoString platform B) Log₂ fold change values were calculated and genes with significant (p<0.05 or p<0.1) differences are shown along with associated immune-related pathways. C) Pathway scores were calculated with nSolver advanced analysis and compared between pre-and post-treatment samples. D) Immune cell abundance based on gene expression signatures were compared between individual patient samples before and after therapy.

Mast cell directed therapy impacts CP/CPPS symptoms.

We next evaluated the efficacy of the therapeutic regimen on CP/CPPS symptoms as determined by the NIH-CPSI, a self-administered validated prostatitis symptom questionnaire. Patients completed the questionnaire at the beginning of the study and weekly till the end of the study. We analyzed the change in the total score as well the individual sub-domains including pain, urinary symptoms, and quality of life. In comparing the pre- to post-treatment, the total score was significantly reduced (p<0.001) with a mean of difference 5.2±1.1 (Fig. 3A). 15 of 20 patients (75%) showed a reduction and 9 patients showed a clinically significant 6 point or greater reduction in the NIH-CPSI total score. The remaining 5 patients showed no change or an elevation in the total score from pre-treatment levels. We also evaluated the weekly symptom questionnaires and observed a significant decreasing trend for total scores over the 3-week treatment period (Fig. 3B). In examining the individual sub-scores, we observed a significant reduction in the pain sub score (p<0.001) with a mean difference of 3.05±0.76, a significant difference in the urinary sub-score (p<0.001) with a mean difference of 1.0±0.34 and in quality of life (p<0.01) with a mean difference of 1.15±0.32. These results show that mast cell directed therapy can have a positive impact on patient symptoms in CP/CPPS.

Figure 3. Mast cell directed therapy reduces NIH-CPSI scores.

A) NIH-CPSI total scores in patients who successfully completed the treatment trial. Groups were compared using ratio paired two t-tailed t with significance shown at p<0.001. B) Weekly change in the total scores in patients under treatment. Data were analyzed by fitting a mixed model and a test for linear trend was performed with p<0.001.

C-E) Pain, Urinary symptoms and Quality of life (QoL) sub scores of the NIH-CPSI in patients before and after treatment analyzed as in A).

Mast cell directed therapy impacts urinary voiding symptoms in CP/CPPS.

We examined urinary voiding symptoms in CP/CPPS patients using the self-administered AUA-Symptom index questionnaire. Like the NIH-CPSI, patients completed the questionnaire at the beginning and end of the treatment period and weekly during the study. Symptom scores at the outset varied widely between patients, with scores in the mild range (0-7) in 8 patients and in the moderate to severe range (≥8) in the remaining 12 patients. We observed a statistically significant reduction in the symptom scores (p<0.05) following treatment with an average difference of 2.95±0.88. As before, we observed a significant decreasing trend for voiding symptom scores over the three-week treatment period. Interestingly, in patients with mild symptoms 3 out of 8 (37.5%) showed a clinically appreciable 3-point reduction in the AUA-SI while in patients with moderate to severe symptoms 9 out of 12 (75%) showed the same response. These results show that mast cell directed therapy has the potential to positively impact the presence of voiding symptoms in CP/CPPS patients.

DISCUSSION

CP/CPPS is an intractable condition that has an uncertain pathogenesis and is treated with empirical therapies that are largely unsuccessful. In this study, we focused on identifying and isolating a specific subgroup of CP/CPPS likely to have a pathogenesis that involves mast cell dysfunction. This effort builds on animal modeling work from ours and other groups that have shown over the years that mast cell dysfunction can lead to some of the symptoms of CP/CPPS such as pelvic pain and voiding dysfunction ^{3, 6, 12, 13}. We had also previously shown in preliminary studies that EPS from men with pelvic pain and voiding dysfunction had appreciably higher levels of mast cell tryptase than healthy controls ⁶. In this study we chose to use a pre-specified criterion of an elevated level in mast cell tryptase as a way of focusing our pilot therapeutic approach to the patients most likely to have benefit from mast cell therapeutic targeting. In using this biomarker-based phenotyping approach, we agree with studies that have described enhanced effectiveness of therapies in CP/CPPS when clinical phenotyping approaches are utilized to categorize CP/CPPS subgroups for subsequent therapeutic approaches ¹⁴. Interestingly, we observed over 80% of CPPS subjects showed a level above our threshold cutoff suggesting that mast cell dysfunction is not uncommon among patients. The cutoff of 25ng/ml utilized is comparable to the normal total tryptase concentrations of 1-15ng/ml in serum that have been reported in studies of mast cell associated conditions such as systemic anaphylaxis and systemic mastocytosis ¹⁵. Currently, we do not yet have data establishing the specificity and sensitivity of the tryptase test for its use as a biomarker for clinical diagnosis in CP/CPPS. The results show that the mast cell tryptase test was able to accurately identify patients with mast cell dysfunction and track the progress of therapy in individual patients.

Mast cell directed therapy was successful in meeting the primary endpoint of a significant reduction in tryptase concentrations in EPS. These studies were informed by earlier work done by our group in an animal model of CP/CPPS where two therapeutic agents – cromolyn sodium and cetirizine dihydrochloride were individually ineffective but demonstrated synergistic efficacy in reducing suprapubic tactile allodynia in the EAP model ³. The combination treatment may directly affect the ability of the mast cells to produce and release cytokines upon activation and degranulation ^{16, 17}. In addition, the administration of the histamine 1-receptor antagonist is known to exert an immunomodulatory function to skew the T cell immune balance in the diseased prostate and dampening the production of cytokines CD4⁺ T cells ¹⁸. The reduction in tryptase concentrations in 15 out of 19 subjects suggests a significant response to therapy. It is noted however, that the drop in tryptase concentrations do not approach the pre-specified control thresholds, suggesting that there is room for improvement in the duration or dosing of the therapeutic regimen. It was also noted that up-to 20% of patients did not accurately complete the study due to various reasons including adherence issues suggesting that measures to reduce obstacles to study adherence may improve patient compliance with the therapeutic regimen. Similarly, we do not know the reasons underlying the 4 non-responder patients in this study and can only speculate that this reflects either underlying biological variability and/or patient specific factors.

Mast cell targeting demonstrated an unexpected and appreciable reduction in immune activation in cells isolated from the post-prostatic massage urine. This modulation of the immune response was noticeable as a reduction in abundance of leukocytes (CD45+) and innate immune cells including macrophages and neutrophils. The adaptive immune pathways that showed a reduction include the Th1 and Th17 pathways that have been previously implicated as driving pain pathways in models of ^{19, 20}. The mechanisms underlying these modulatory responses remain to be identified, but the pronounced reduction in multiple innate and adaptive immune pathways suggest a central role for mast cells in driving the inflammatory response in the prostate environment.

The impact of mast cell targeting on symptoms was significant with positive benefit seen in reducing pain, urinary symptoms, and quality of life scores. These changes, moreover, showed a decreasing trend with increased duration of treatment showing enhanced benefit. As before, the absence of response in some patients reflects the heterogeneity of the patient therapeutic response. The small sample size of these studies also limited our ability to establish a direct correlation between reduction in tryptase concentrations and the symptom response. We also observed these positive trends using the AUA-SI where it was noted that the presence of voiding symptoms was widely variable at the outset. Patients starting with moderate to severe symptoms showing a higher likelihood of demonstrating clinically significant benefits. Meta-analysis of clinical trials in CP/CPPS have shown that for every additional week of any given treatment, total NIH-CPSI score decreased by an average of 0.19 points and implied a requirement of 32 weeks of treatment to achieve a total NIH-CPSI reduction of 6 points ²¹. Given these sobering facts from previous trials, our data showing a statistically significant reduction in symptoms, the downward trend in scores with increasing time and achievement of clinically significant effects in an appreciable number of patients in a 3-week treatment window is notable. Caveats to this include the fact that this pilot study was not a randomized control study and could not account for placebo-related effects.

CONCLUSION

This pilot study presents a method to identify CP/CPPS patients with mast cell dysfunction and target them for therapeutic intervention using a novel, safe combination therapeutic approach ²². The resulting subgroup of CP/CPPS patients were shown to have a therapeutic response as measured primarily by a reduction in mast cell tryptase, potential surrogate biomarker and reduction in CP/CPPS symptoms.

Figure 4. Mast cell directed therapy reduces AUA-SI scores.

A) AUA-SI scores from patients who successfully completed the treatment trial. Groups were compared using ratio paired two t-tailed t with significance shown at p<0.05. B) Weekly change in the total scores in patients under treatment. Data were analyzed by fitting a mixed model and a test for linear trend was performed with p<0.001.