The long-standing debate about the role of inflammation in the pathology of Interstitial Cystitis/ Bladder Pain Syndrome (IC/BPS) has now attracted the attention of artificial intelligence and machine learning tools. On that count, use of the machine learning tool CIBERSORT on bladder tissue specimens of IC/BPS patients identified immune cell infiltration with negative urine culture report as the defining feature of IC/BPS. While the 85% response rate of cyclosporine in a subset of IC/BPS patients validates the critical role of inflammation in IC/BPS , vital organ toxicity of cyclosporine  motivates the search of safer alternatives like Suplatast tosilate,  an oral anti-inflammatory drug of Japan that targets type 2 T-helper cells and used on IC/BPS patients.  
There is a growing chorus for drugs from natural sources as the prototype of non-steroidal anti-inflammatory (NSAIDs)- Salicylic acid was derived from the bark of willow tree (L. Salix), which is characterized by multiple actions including the inhibition of the enzyme cyclooxygenase (COX). However, Salicylic acid possess poor risk-benefit ratio for IC/BPS patients owing to the association of NSAIDs with stomach ulcers and salicylic acid is shown to promote cystitis in dogs (Am J Vet Res. 1986;47(12):2621-3.).  Therefore, past experience justifies the investigations into a well-known folk remedy of Native Americans, obtained from the bark of white birch (Betula alba), whose active moiety is Betulinic acid, three times heavier in molecular weight than the popular congener (sibling)- Salicylic acid.  Betulinic acid is undergoing clinical testing as a topical ointment against melanoma (ALS 357)  in a Phase I clinical study  (NCT00701987) owing to the ability of Betulinic acid to induce apoptosis through changes in mitochondrial membrane potential and the production of reactive oxygen species (ref.1). While the action of Betulinic acid on mitochondria
 could also exert anti-inflammatory effect (ref.2), the self-assembly of the pentacyclic triterpenoid structure into different sizes and structures could entrap other potent anti-inflammatory drugs for synergistic action or earn the moniker "vehicle with benefits". Hence, we examined the selectivity of Betulinic Acid for immune cells over urothelium and the potential therapeutic advantage of Suplatast tosilate entrapped in Betulinic Acid nanospheres over free Suplatast tosilate.

Human Jurkat T cells, and mouse urothelial cells TRTU-H1 cells procured from ATCC  were seeded at a density of 5000 cells/well in 96 well plates 18h before treatment with Betulinic Acid, Suplatast or Betulinic acid nanospheres encapsulating Suplatast. Jurkat T cells were cultured in RPMI for with 10% FBS whereas for TRTU-H1 cells were cultured in KSFM with 1% antibiotic solution of Penicillin, Streptomycin and Amphotericin B, followed by maintenance at 37ºC in a humidified atmosphere of 5% CO2. The cells were treated with concentrations ranging from 0.1 to 10 µM of Betulinic Acid and free Suplatast and Betulinic acid nanospheres encapsulating Suplatast.18h later, 50 µl MTT solution at a concentration of 0.5 mg/ml was added to the cells for four hours followed by the addition of DMSO and 30% SDS lysis solution in the ratio of 90:60 to dissolve the formazan crystals. The absorbance was measured at 570nm to determine the half maximal inhibitory concentration (IC50) - the concentration required to achieve 50 % decrease in cell viablity.

The concentration response curve in the attached figure measures potency by IC50 - concentration required for decreasing viablity by  50%, and a right ward and leftward shift in IC50 indexes an decrease and increase in potency, respectively of Betulinic acid and Suplatast tosilate on Jurkat T- cells and TRT-HU1 cell lines. Two graphs display the differential impact of Betulinic acid encapsulation on the potency of Suplatast tosilate as the potency of Suplatast tosilate on Jurkat T- cells and TRT-HU1 cell line, representing urothelium was increased and decreased, respectively. The differential impact of Betulinic acid appears to be linked to the doubling time as Jurkat T- cells doubled at a faster rate (20 h) than TRT-HU1 cells (42 h). The faster doubling time of immune cells is bound to increase the demand of nutrients and lower the specificity of nutrients and the misperception of Betulinic acid as a nutrient is linked to its cytotoxic effect on rapidly dividing Jurkat T- cells mimicked the sensitivity of cancer cells to Betulinic acid, whereas TRT-HU1 cell line for resident urothelium could tolerate much higher concentrations of Betulinic acid and nanospheres encapsulating Suplatast tosilate. Indeed, the encapsulation of Suplatast by Betulinic acid generated a ten fold difference in cytotoxicity of Suplatast between immune and urothelial cells to 144 fold.

The obvious similarities in the response of immune cells and cancer cells to Betulinic acid incriminates an overlap in the metabolic reprogramming of immune and cancer cells to facilitate cell proliferation in nutrient-poor environments of inflammation and tumor, respectively (ref.3). The consumption of nutrients to fuel faster cell divison is bound create a scarcity of nutrients and trigger metabolic signals of pseudo-starvation which in turn will induce epigenetic changes to re-route other metabolic pathways or metabolic reprogramming to fuel growth with alternative nutrients. In that context, Betulinic acid mimics the structure of cholesterol, a nutrient (ref.1-2) and like cholesterol, Betulinic acid also preferentially accumulate in mitochondria of immune and cancer cells.  Mitochondrial accumulation of Betulinic acid in fast growing immune cells is likely to induce apoptosis through changes in mitochondrial membrane potential, production of reactive oxygen species, and the opening of mitochondrial permeability transition pores, that results in the release of mitochondrial apogenic factors, activation of caspases, and DNA fragmentation. Thus, metabolic reprogramming of T cells significantly enhances the uptake of Betulinic acid nanospheres which causes higher cytotoxicity of Betulinic acid on immune cells relative to cell line of urothelium. The infiltration and proliferation of immune cells in bladder wall of IC/BPS patients is critically dependent on metabolic reprogramming can be inferred from the constrained supply of nutrients and oxygen ensuing from venous congestion on bladder biopsy specimens of IC/BPS patients. 
The metabolic vulnerability of immune cells underpins their selectivity for Betulinic acid and may explain the reports on liver fibrosis suppression by Betulinic acid in treatment of non-alcoholic steatohepatitis which bodes well for the treatment of bladder wall fibrosis. Though Betulinic acid may not be as potent anti-inflammatory as salicylic acid or other NSAIDs but the larger molecular size of Betulinic acid fosters self-assembly into nanospheres for encapsulation of Suplatast tosilate and offer opportunities for synergistic action after intravesical administration as the toxic effects of Suplatast tosilate on urothelial cells were reduced upon entrapment in Betulinic acid nanospheres. Thus, Betulinic acid could be a "vehicle with benefits" capable of reducing the reported toxicity of Suplatast tosilate in IC/BPS patients.

Here, we report that clinically viable, Betulinic acid obtained from Birch tree likely confers metabolic signals of pseudo-starvation to faster dividing immune cells by selectively accumulating in mitochondria, which could be valuable in down-regulating autoinflammatory responses including fibrosis associated with IC/BPS. In addition, the self-assembling property of Betulinic acid is worthy of the designation, "vehicle with benefits" for Suplatast tosilate.  The similar response of cancer and immune cells to Betulinic acid alludes to a metabolic vulnerability conferred by faster cell division which explains the frequent duplication of anticancer drugs as anti-inflammatory agents: Methotrexate, BCG, Imatinib mesylate (Gleevec).

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