The definitive characteristic of hemorrhagic cystitis is moderate to severe inflammation of the bladder wall that is unresponsive to conventional steroidal anti-inflammatory drugs and systemic immunosuppressants. When administered systemically, these drugs create a downhill concentration gradient from the bloodstream to the bladder, which can lead to drug-related toxicity affecting vital organs such as the liver or kidneys. The potential for toxicity is higher than the potential benefit of increased drug efficacy in the bladder as bladder gets the lowest concentration of administered drug.
On the other hand, intravesical administration follows the principle of "Bladder before Blood (BBB)" by reversing the typical concentration gradient of systemic administration. Previous studies on intravesical chemotherapy for bladder cancer have demonstrated that the bladder mucosa receives the highest concentration of the drug, with the concentration gradient driving diffusion from the mucosa to the blood resulting in exponentially lower concentrations in the bloodstream (ref. 1). However, the exponential difference between bladder and blood concentrations of the drug in non-cancerous disease conditions has yet to be determined.

Tacrolimus is a potent macrolide calcineurin inhibitor that exerts an anti-inflammatory effect. However, its insolubility in water presents a challenge for intravesical delivery. Tacrolimus has been encapsulated in liposomes for intravesical administration in rodent models, and urine and blood levels of tacrolimus after instillation have conformed to the BBB principle (ref. 2-3). Here, we report on clinical translation with pharmacokinetic evidence collected from 13 subjects with refractory moderate to severe hemorrhagic cystitis who were administered escalating doses of intravesical liposomal tacrolimus (LP-10).
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A prospective, multi-center, dose-ranging study was conducted on adult male and female subjects (n=13) with refractory moderate to severe hemorrhagic cystitis, as determined by a physician. The age range of the subjects was 25 – 89 years, average age 67 years. The subjects were sequentially assigned an escalating dose of LP-10 (2, 4 and 8mg) dispersed in 40mL, administered via urethral catheter, with instillations separated by more than 3 days but less than 7 days.
To determine the pharmacokinetic profile of LP-10, urine and blood samples were collected from each subject before dosing and at 0.5h, 1h, 2h, 4h, and 48h after the administration of LP-10. Standard methods were used to analyze the collected samples for tacrolimus levels, and the geometric means ± standard deviation of peak blood and urine concentrations at each dose level were computed.

The thousand-fold difference in the y-axis scale for the left and right panel of Fig.1 depict the microgram per mL range of tacrolimus in collected urine samples as opposed to nanogram per mL range for tacrolimus in blood at all time points, respectively. The log scale difference in the urine and blood levels of tacrolimus after intravesical administration of LP-10 (Fig.1) is a testament of  downhill concentration gradient from bladder mucosa to blood in conformity with the BBB principle.  Since past animal studies validated a linear relationship between the drug concentration in urine and the drug concentration in bladder tissue (ref.1), urinary tacrolimus concentration of treated human subjects in microgram range is a non-invasive surrogate for the tacrolimus concentration delivered to the bladder mucosa of human subjects without relying on the biopsy-based tissue analysis after intravesical administration of LP-10. Accordingly, a dose-dependent increase in mucosal concentration of tacrolimus is projected from a dose dependent increase in the geometric mean peak concentration (Cmax) of 11.39 ±1µg/mL to 9.783±6.973 µg/mL to 31.959 ±5.20 µg/mL in urine sample collected at 0.5h after instillation of 2mg , 4mg and 8mg dose, respectively( left panel of Fig.1).  Urine tacrolimus levels dropped drastically at 2 and 4 hours post-instillation and became undetectable by 48 hours.  In contrast, geometric mean Cmax in blood at all dose levels was reached 1h after administration(right panel of Fig.1) and Cmax nearly doubled from 2.683±1.14ng/mL to 4.266±1.37ng/mL for 2mg and 4mg dose, respectively and reached 3.731±1.611ng/mL with 8mg dose. Thus, the urine and blood tacrolimus levels conform to the BBB principle of intravesical route and assure reduced systemic exposure and increased likelihood of efficacy over toxicity.  Blood levels always remained within the safe range (< 15 ng/mL), and the lower limit of detection by standard assay is 1.5 ng/mL.

Topical tacrolimus is a clinically proven  anti-inflammatory drug for localized treatment refractory inflammatory diseases of skin and mucous membranes. Past studies (ref.2) reported the promise of liposomes as a suitable delivery platform  for intravesical delivery of tacrolimus in a rat model of radiation cystitis and cyclophosphamide hemorrhagic cystitis.  Here, we report the clinical translation of the same approach in a multi-center clinical trial on patients diagnosed with refractory moderate to severe hemorrhagic cystitis. Liposomes achieved the downhill concentration gradient from urine to blood of tacrolimus in compliance with the BBB principle with a dose dependnent rise in Cmax of blood. Liposomes are also biocompatible with injured urothelium and known to deliver drugs via endocytosis,  whereas clinical studies similar to ref.3 used organic solvents, either  a mixture of hydrogenated castor oil and alcohol or 10- 50% DMSO as vehicle for water-insoluble tacrolimus. The instillation of organic solvents as vehicle for tacrolimus may not be safely tolerated by patients suffering from refractory moderate to severe hemorrhagic cystitis and organic solvents are also likely to aggravte the bladder injury and confound the therapeutic effect of tacrolimus from alcohol and DMSO.  Unlike the Cmax in blood at 1h with LP-10, the blood tacrolimus levels after intravesical administration in DMSO were reported at 30min in interstitial cystitis patients., which could be related to the injury to the bladder mucosa. Previous studies confirmed that compared to liposomes, use of organic solvents as vehicle of tacrolimus increased the systemic exposure of tacrolimus after intravesical administration (ref 2). Compared to biocompatiblity of liposomes, organic solvents were found to be toxic on bladder mucosa. Volume of instillation for LP-10 was similar to previously reported studies in human bladder (ref.3). Liposomes assist in the adherence of water-insoluble drug with tight junctions and then higher lipophilicity of tacrolimus aids in navigating the tortuous gap in tight junctions. Taken together, the intravesical absorption of LP-10 (lipsomsal tacrolimus) in rodent and human bladder mirrors the kinetics for intravesical absorption of paclitaxel in bladder cancer patients.

Intravesical administration of LP-10 complies with BBB principle and achieves dose dependent downhill concentration gradient of tacrolimus from bladder to blood by inverting the downhill concentration gradient from blood to bladder erected by systemic administration of tacrolimus. While organic solvents are toxic to bladder mucosa, liposomes are demonstrably safe and tolerable for subjects with refractory moderate to severe hemorrhagic cystitis and LP-10 is certainly safer than agents used for intravesical sclerotherapy : alum, silver nitrate or formalin.

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Continence 7S1 (2023) 100824
DOI: 10.1016/j.cont.2023.100824