Chronic visceral pain disorders, such as interstitial cystitis/bladder pain syndrome (IC/BPS), are among the most difficult types of pain to treat and response to treatment is often negligible.  Evidence suggests that oxidative stress and inflammation may play important roles in the pathophysiology of a number of chronic pain disorders.  The enzyme purine nucleoside phosphorylase (PNPase) is important for the metabolism of ‘tissue protective’ purines to ‘tissue-damaging’ purines that generate free radicals (e.g., reactive oxygen species or ROS).  Our preliminary studies reveal that inhibition of PNPase (oral treatment with 8-aminoguanine or 8-AG) yields significant improvement in a bladder-centric model of IC/BPS (cyclophosphamide, CYP). The aim of this study uses the CYP model to validate a non-opioid based target, namely PNPase for the treatment of IC/BPS.

Adult Sprague Dawley rats were divided into the following groups: 1) control; 2) CYP (75 mg/kg/i.p. every 3 days; sacrificed day 8) and CYP plus a PNPase inhibitor, 8-AG (5 mg/kg given orally in drinking water starting 14 days prior to start of CYP). The Institutional Animal Care and Use Committee approved all procedures. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).  We utilized metabolic cage to assess bladder function and behavioral assessment (von Frey microfilaments) to assess mechanical allodynia. After sacrifice, urinary bladders as well as lower lumbo-sacral spinal cords were collected, and tissues were assessed for gross visual inspection for inflammation and biomarkers for pain/inflammation (using immunohistochemistry and western immunoblotting).

We find a significant increase in voiding frequency (190% +/- 18% fold increase) and a decrease in the intercontraction interval (ICI) (70% +/- 9% fold decrease) in rats chronically treated with CYP which also induces a bladder inflammation with increased petechiae hemorrhage in the bladder wall (assessed macroscopically). We also find increased behavioral response in CYP rats to mechanical stimuli (80% +/- 4% fold decrease).  Both bladder voiding dysfunction and tactile allodynia in addition to the appearance of gross inflammation were restored to control levels by treatment with 8-AG. 
     Similar to IC/BPS patients, CYP-rats exhibit elevated levels of the cytokine interleukin-1B (IL-1B), monocyte chemoattractant protein-1 (MCP-1) and toll-like receptor 4 (TLR-4, 150% +/- 10%-fold increase).  8-AG treatment of CYP rats completely prevents the increased expression of these pain biomarkers as compared to controls.  In addition, we find that CYP-rats are associated with increased spinal cord activation of microglia (160% +/- 20%  fold increase; measured by densitometry) and increased expression of IBA-1 (200% +/- 30% fold increase), a calcium binding protein expressed in microglia. These changes are both normalized by treatment of CYP-rats with 8-AG.  PNPase is highly expressed in microglia, as compared to astrocytes and neurons.

Our preliminary findings reveal altered outcome measures in a CYP-rat model for bladder pain/inflammation.  These include: 1) bladder voiding dysfunction, 2) enhanced pain behavior, 3) elevated biomarkers associated with visceral pain (IC/BPS) that have been observed in patients and 4) alterations in spinal cord microglia (which have a high PNPase content).  In addition, inhibition of PNPase (with 8-aminoguanine, 8-AG) restores many of the functional, behavioral, morphological and structural alterations in the IC/BPS animal model to a control state.

Emerging evidence has revealed that alterations in the enzyme purine nucleoside phosphorylase (PNPase) reflects the participation of oxidative injury and cellular damage.  PNPase products generated either within the target lower urinary tract (LUT) cell or remotely could damage LUT cells via ROS production.  In sum, our preclinical findings support the use of PNPase inhibitors as a new class of drug therapy with corrective and restorative actions at the cellular level to improve bladder structure and function and reduce pain behavior and inflammation in a bladder-centric animal model for IC/BPS. Thus, while IC/BPS can stem from multiple causes making a single drug ineffective, we believe that targeting a single enzyme, PNPase, will restore the purine dysregulation thereby reducing inflammation and free radical formation while increasing antioxidant capacity.