Development and investigation of a novel device with gemcitabine for hyperthermic intravesical chemotherapy

Abstract Purpose To evaluate the safety and efficacy of a novel hyperthermic intravesical chemotherapy (HIVEC) device in combination with gemcitabine. Materials and methods A pilot clinical trial was performed on patients with high-risk non-muscle invasive bladder cancer (NMIBC), who received HIVEC via the novel device (BR-PRG). Treatment regimen included eight weekly instillations of intravesical GEM (3 g in 150 mL normal saline [NS]) at a temperature of 45 °C for 60 min. Assessment of adverse events (AEs) was the primary objective of the trial. Disease recurrence and the thermal stability of GEM were also analyzed. Results A total of 116 HIVEC treatments were delivered. Fifteen and eighteen patients were included in the effectiveness and safety analysis, respectively. Median follow-up was 12 months; five patients experienced a disease recurrence. One-year cumulative incidence of recurrence was 23.8% in EORTC intermediate risk group and 37.5% in high-risk group. Ten patients experienced at least one AE, with the most common being acute urinary tract infection, followed by urinary tract pain, and hematuria. Two patients experienced acute cystitis (grade 3 AE) and instillations were postponed until full recovery. Other AEs were minor, and no systemic toxicity was observed. The contents of GEM in solution of 0.9% NS or NS mixed with artificial urine were stable at 25 °C, 37 °C, 43 °C, 45 °C, 47 °C and 50 °C for 2 h. Conclusion GEM can be an ideal drug for use in HIVEC due to its good thermal stability. BR-PRG, combined with GEM was safe and effective in administering HIVEC.


Introduction
Globally, bladder cancer has been ranked as the fourth most common type of cancer in men and the 11th most common cancer in women [1].More than 75% cases are diagnosed as non-muscle invasive bladder cancer (NMIBC).Transurethral resection of bladder tumors (TURBT) is the standard treatment for early NMIBC [2].However, the local control rate of this disease is relatively low, with up to 45% patients experiencing recurrence within the first year after TUBRT alone.In addition, progression in tumor grade and stage may also be observed [3].Intravesical Bacillus Calmette-Gu erin (BCG) therapy is the preferred treatment for reducing the recurrence rate or progression in intermediate-and high-risk NMIBC; however, limited availability and toxicity of BCG impede its clinical application.In recent years, hyperthermic intravesical chemotherapy (HIVEC) has been reported to enhance the efficacy of regular intravesical chemotherapy and to benefit BCG unresponsive patients [4].Currently, radiofrequencyinduced hyperthermia (RITE) and conductive hyperthermic chemotherapy, which are performed using the Combat Bladder Recirculating system (BRS) or the Unithermia system are primary techniques utilized in the application of HIVEC [5].Unfortunately, none of the above-mentioned devices are available in China, though the BR-TRG-I urinary bladder hyperthermia treatment system, which is similar to the BRS is widely used [6,7].This system, which was originally designed for hyperthermic intraperitoneal perfusion chemotherapy has some shortcomings when used for bladder perfusion, including a relatively low drug concentration, an expensive disposable perfusion pipe and a massive size [8].Therefore, we developed a new device for HIVEC owing to our experience of designing BR-TRG-I and long-term clinical practice of local hyperthermia, to better satisfy the clinical needs and improve the perfusion efficiency of HIVEC.
Gemcitabine (GEM), as the recommended drug for post-TURBT intravesical chemotherapy, has received extensive attention because of its less adverse side-effects, better tolerance and wide anti-tumor spectrum.Due to its enhanced antitumor effect in hyperthermia, it is considered as the preferred chemotherapy drug for HIVEC [9,10].GEM administrated in HIVEC should be heated to a target temperature and maintained for a dwell time of 60 min.To the best of our knowledge, the stability of GEM in relatively high temperature is still unknown.Besides, during HIVEC, 100-150 ml urine is produced into the bladder and the effect of urine on drug stability is also unclear.Thus, the stability of GEM at different temperatures and in different menstruums was investigated in the present research.
The combination of heat and intravesical chemotherapy has been utilized both in the neoadjuvant (pre-TURBT) and adjuvant settings (post-TURBT), with promising results in terms of efficacy, safety and tolerability, when used in variable device-assisted technologies.However, treatment of high-risk NMIBC (HRNMIBC), especially carcinoma-in-situ (CIS) and BCG-unresponsive cases is still controversial.As a result, it is crucial to develop new HIVEC treatment regimens, hyperthermia devices, and instillation chemotherapy agents.
Herein, we conducted a clinical trial on patients with HRNMIBC, by applying our new urinary bladder hyperthermia treatment system with GEM as the chemotherapeutic drug.Furthermore, we assessed the thermal stability of GEM, and characteristics as well as safety of the proposed novel urinary bladder hyperthermia treatment system.

Patient selection
From July 2019 to September 2021, patients with HRNMIBC were recruited from the Guangzhou Medical University affiliated Cancer Hospital for a pilot clinical trial of HIVEC using the new device.The trial was approved by the ethics committee of the hospital and registered (chictr.org.cn:ChiCTR1900022099).Signed informed consent was obtained from all patients prior to enrollment.All cases underwent immediately intravesical instillation therapy with GEM, within 24 h of TURBT.Inclusion criteria were as follows: age >18 years, Eastern Cooperative Oncology Group performance status <2, life expectancy >24 months, and a negative pregnancy test for women with childbearing potential.HRNMIBC was defined by Chinese guideline including low grade of stage T1 tumors and high grade or CIS with any stage disease [11].Included patients were reclassified according to the European Association of Urology NMIBC guidelines [2].The exclusion criteria were as follows: clinical stage !T2, nonurothelial histology, previous history of upper urinary tract tumors, known GEM allergy, active urinary tract infection, severe bleeding disorder, bladder volume <150 ml, urethral stricture impeding 18 F catheterization, partial cystectomy, previous pelvic radiotherapy/systemic chemotherapy, previous intravesical chemotherapy, uncontrolled significant medical or psychiatric disease, concomitant malignancy at a different location and known vesico-ureteral reflux.

Transurethral resection of bladder tumor
All patients underwent TURBT under general anesthesia, performed by two surgeons in our hospital.The standard bipolar resection technique was used in every case.Tumor size, multifocality, and location were recorded.All visible tumors were resected, and deep bladder wall biopsies were taken.
Additionally, systemic mapping bladder biopsies were also obtained.Thereafter, all specimens were sent for a pathological review.All patients received routine indwelling threeway Foley catheter and continuous bladder irrigation was done using large amounts of 0.9% saline at room temperature until the urine turned clear.HIVEC was implemented within three days of TURBT, if there was no gross hematuria.

Characteristics of the new urinary bladder hyperthermia treatment system
This novel HIVEC device, named as the BR-PRG (Figure 1(A,B)) was developed on the basis of BR-TRG-I type highprecision intraperitoneal thermal perfusion treatment system which was independently created at our center and has been approved for the treatment of malignant ascites and peritoneal cancer due to its safety and efficacy [12].Despite using the same technology of conductive hyperthermic chemotherapy, the BR-PRG differs from BR-TRG-I in a few aspects.First, the fluid is heated externally through electromagnetism.Second, to reduce the fluctuations of the treatment curve and make it more stable, the metal container of the device is designed to have two chambers: inner one for the input of chemotherapy drug and the outer one for the liquid used for heat conduction (Figure 2(A)).Third, the device has a disposable light-proof tube which can be connected with the 18 F three-way Foley catheter located in the bladder to form a closed circulation system (Figure 2(B)).A continuous circulation perfusion system is created by driving a peristaltic pump and siphoning liquids from different levels.Meanwhile, the adoption of an automatic lift bracket simplifies tube assembly on the top and improves the efficiency of siphon at the bottom.Fourth, the integrated function of monitoring real-time urine volume is realized by continuous measurement of the increasing weight of the drug container.Furthermore, with detectors on the tube, the volume of fluid in the bladder can be calculated to maintain moderate filling of the bladder and prevent overfilling induced adverse events (AEs) associated with HIVEC.Lastly, the compact size, added safety features and intelligence software enable precise control of HIVEC and ensure the safety of the treatment (Figure 3(A,B)).

Thermal stability study of gemcitabine hydrochloride
To assess the stability profile of intravesical GEM under varying temperature conditions and urinary pH, we prepared a reference solution (GEM 3 g dissolved in 0.9% normal saline solution [NS] 150 ml) and a test solution (GEM 3 g dissolved in and NS 150 ml mixed with artificial urine (AU) solution 150 ml).Both the solutions were examined for stability at the temperatures of 25 C, 37 C, 43 C, 45 C, 47 C for two hours.Thereafter, the pH, clarity, visible particles, related substances and contents of GEM were determined according to the Chinese Pharmacopeia (version 2020).The amounts of the related substances were calculated and expressed as a percentage of GEM, as described previously [13].

Treatment schedules
HIVEC achieved with the BR-PRG device was performed within three days after TURBT.The treatment schedule consisted of six to eight weekly administrations of HIVEC, followed by 10 monthly intravesical instillations of GEM alone.HIVEC was administered at a concentration of 3 g GEM diluted in 150 ml of sterile saline, heated to a target temperature of 45 ± 0.5 C, and maintained for a dwell time of 60 min.The constant liquid perfusion rate was set as 50-150 ml/min.While the subsequent treatment regimen consisted of 1 g GEM diluted in 50 ml of sterile saline at room temperature and maintained in bladder for 60 min in a different position.Treatments were discontinued if severe  side-effects or GEM allergy developed, the patient withdrew consent, or the physician deemed it in the patient's best interest.

Trial design and end points
The primary objective of this pilot study was to determine the safety and heating efficiency of the novel BR-PRG device [14].The study design required 15 evaluable patients [15].The primary study end point was the occurrence of AEs, which were defined as any adverse changes in health or side effects experienced by the participants during HIVEC or follow-up.Routine blood tests and urinalysis were carried out prior to each HIVEC treatment.The white blood cell (WBC), hemoglobin, platelet counts in blood and leucocyte, erythrocyte in urine were recorded, WBC count < 4.0 Â 10 9 /L was estimated as bone marrow suppression.AEs recorded prior to, after every HIVEC treatment and at each follow-up visit were graded according to the Common Toxicity Criteria of the National Cancer Institute for AEs (CTCAE), version 4.0.In the case of side effects, dose modifications were not allowed; instead, treatment was delayed.Follow-up was performed after completing TURBT.Cystoscopy, urinary cytology and imaging examinations of the urinary tract were repeated every three months for two years.Recurrence or progression was pathologically confirmed.Meantime, the one-year cumulative incidence of recurrence for each European Organization for Research and Treatment of Cancer (EORTC) risk group for recurrence was also analyzed.

Statistical methods
Standard descriptive summary statistics were calculated, and continuous variables were summarized as medians with their range or interquartile range (IQR).The tumor recurrence risk was estimated via the cumulative incidence function.95% Confidence intervals (95%CIs) for these endpoints were determined using greenwood method.All statistical analyses were performed using SPSS statistical software (version 24; IBM Corporation, Armonk, NY, USA) and R version 4.2.0 with the following packages installed: foreign, survival, ggplot2 and survminer.

Results
In general, GEM demonstrated ideal solubility and stability at the concentration of 3 g in 150 ml solution (20 mg/mL).The solution was always clear, colorless and transparent.The content of GEM was constantly stable at 25 C, 37 C, 43 C, 45 C, 47 C and 50 C for 2 h, even after addition of AU (Figure 4).The urinary pH did not affect the stability of GEM.The total content of GEM was between (98.85 ± 0.75)% and (100.65 ± 1.16)% (RSD 1).According to the specifications of the Chinese Pharmacopeia, the impurity content of cytosine should not exceed 0.1%, that of a -anomer should be not more than 0.1%, that of any other impurity should be not more than 0.1% individually, and the sum of all impurities should not exceed 0.2%.In our study, under varying temperature conditions and urine pH, cytosine concentration was (0.000 7 ± 0.000 08) %, a-isomer was undetected, each of any other impurity was (0.03 ± 0.009)%, and the sum of all impurities was less than (0.04 ± 0.03)%, all of which met the requirements of the Chinese Pharmacopeia.Good thermal stability of GEM signified that it rarely decomposed to substances that might be harmful to the patient.
A total of 15 patients were enrolled in this preliminary clinical trial.While three patients did not complete at least five HIVEC instillations under the pressure of the COVID-19 pandemic and withdrew from the study, three additional local patients were enrolled to make up for the loss.Therefore, 15 patients were included in the effectiveness analysis, while 18 patients were included in the safety analysis.Median follow-up was 12 months (range: 30 and IQR: 9 À 32) and the last enrolled patient was followed for more than six months.Patient and index tumor characteristics are described in Table 1.
During the follow-up period, five patients experienced disease recurrence at 3, 6, 9, 12 and 32 months, respectively, and underwent a second TURBT.Three of the five patients had recurrent tumor of the same stage as the primary tumor (n ¼ 2 for T1HG, n ¼ 1 for T1 þ CIS), one patient with primary T1 tumor experienced a recurrent CIS, and one had a HG primary with a LG recurrence.The two patients with CIS recurrence underwent a radical cystectomy and the one patient who initially enrolled with CIS experienced no recurrence during the 18-month follow-up period.The one-year cumulative incidence of recurrence were 23.8% (95%CI: 0, 48%) for EORTC intermediate risk (recurrence score 5-9) group, and 37.5% (95%CI: 0, 68%) for high risk (recurrence score 10-17) group respectively (shown in Figure 5(A,B)).
A total of 116 treatments of HIVEC were delivered in this study.All HIVEC treatments were carried out at the target temperature of 45 ± 0.5 C, maintained for one hour, and the median volume of urine produced was 80 ml (range: 23-210 ml).Eighteen patients were included in the safety analysis, three of whom did not complete at least five HIVEC instillations due to the COVID-19 pandemic and not because of severe side effects.The changes in WBC revealed no concerning trends of bone marrow suppression and no serious complications such as bladder contracture or GEM allergy were observed in this study.In 10 patients, at least one AE was reported during the instillation course, whereas the remaining eight patients had no side effects.Table 2

lists all
AEs encountered during the trial.The main AEs were acute urinary tract infection and pain.Two patients experienced acute cystitis during the course of HIVEC and were treated with oral and IV antibiotics, with subsequent instillations delayed for one week to allow complete eradication of the infection.The AEs of urinary tract pain and hematuria were self-limiting and were reported among all the patients with urinary tract infections.

Discussion
TURBT alone is considered to have a limited potential in preventing NMIBC recurrences, as supported by the high variability in the three-month recurrence rate following the procedure [16].It is necessary to consider postsurgical adjuvant therapy in these patients.At present, the most common postoperative treatment is intravesical instillation of chemotherapy or BCG, which has been in use for nearly 30 years.BCG is considered the most superior for prevention of tumor recurrence, but its efficacy is far from optimal because of its relatively frequent side effects and supply shortage.The anatomy of human bladder allows delivery of drugs through a catheter to the target site with minimal local side effects, while avoiding systemic toxicity.Accordingly, different technologies of intravesical hyperthermia have been recently developed and applied to clinical practice.RITE with Mitomycin C (MMC) achieved increased recurrence free survival at 24 months with much fewer side effects compared with BCG, in patients with intermediate-and high-risk NMIBC [17].While this system is effective, it has several drawbacks including very high cost and a significant rate of bladder burn injuries [18,19].To address these issues, the technology of conductive hyperthermic chemotherapy was promoted, and several devices (BRS, Unithermia and BR-TRG-I) based on this technique have been designed, including the novel device (BR-PRG) presented in this study.These devices are far less expensive and do not appear to cause bladder burns.Meanwhile, some issues that may influence the efficiency and tolerance of treatment should not be neglected.It is well known that the bladder wall is folded on itself in the constricted state and the folds disappear when the bladder is fully stretched.The folded mucosal layers present a barrier to effective fluid diffusion [9,20].In the present research, the initial perfusion volume of the BR-PRG device was set to 150 ml, which guaranteed influx of 80-90 ml liquid in the bladder at the beginning of HIVEC in order to decrease the mucosal folds and exclude some liquid in the circulation tube.It was understood that the patient will eventually produce urine and have a full bladder.However, there is a tradeoff regarding the value of bladder filling.On one hand, stretching of the wall will reduce the folded layers, making the mucosal surface more exposed to the drug, but on the other hand, bladder filling will dilute the drug.In this regard, it has been reported that drug concentration is more important than the treatment duration [21].Given that, the HIVEC treatment schedule of BR-PRG utilizes GEM 3 g dissolved in 150 ml saline, this concentration is equal to the regular instillation regimen of GEM 1 g dissolved in 50 ml saline during the initial instillation.After a typical treatment of 1 h, the final concentration of HIVEC would be nearly twice that of the regular instillation group if both produced urine of about 100 ml (3 g/250 ml, 1 g/150 ml), even though HIVEC group produced a little more urine than the regular instillation group.In an early clinical trial involving intravesical MMC and radiofrequency hyperthermia, 82-95 ml of urine was recovered from unheated control patients versus 130 ml recovered from patients who received hyperthermia treatment [22].In our study, the median volume of urine production in each HIVEC cycle was 80 ml, but the range was wide (23-210 ml), and even the same patient produced varied volumes of urine during different courses.The principal influencing factors may be the amount of water intake before the treatment and the degree of anxiety during the course.Though, whether the system of conductive hyperthermic chemotherapy causes increased urine production is still unknown, it is believed that too much urine results in overfilling of the bladder, which is unfavorable for the therapy, especially in the terms of side effects and patient tolerance.By monitoring the increased weight of fluid in the drug container and because of the closed-loop circulation design, BR-PRG could calculate the volume of urine produced and enable suitable adjustments of bladder filling in real time, to allow stretching of the bladder wall, reduce the size and number of folds and avoid vesical rupture or bladder irritation caused by overfilling.BR-PRG was made simple and safe to use by combining hyperthermia with electromagnetism, incorporating safety features and using intelligence software.
The majority of HIVEC treatments performed to date have utilized MMC as the chemotherapy agent.However, the solution of MMC exhibits approximately 5-7% drug degradation when incubated at 50 C for 50 min, and this accelerates under strong acidic and alkaline conditions [23,24].In addition, the solubility point of MMC is only 0.8 mg/mL at room temperature (23 C), which demonstrates that the treatment of 1.0 mg/mL or 2.0 mg/mL used in intravesical therapy should be undertaken with some caution [25].In the present study, GEM proved to have good thermal stability at different concentrations, remaining stable at 25 C, 37 C, 43 C, 45 C, 47 C, 50 C for 2 h, in NS as well as NS mix AU menstruum.In their study, Grimberg et al. noted that the solubility point of GEM was up to 60.3 mg/mL at 23 C and this dosage remained soluble even after two hours of hyperthermia at 43 C and at pH values of 4.0,6.0 and 8.0 [25].The intravesical dose of GEM 20 mg/mL or 40 mg/mL has been reported to be safe and well tolerated, with no more than 1 mg/mL GEM detected in the serum [26].Therefore, GEM  could be an ideal alternative for use in HIVEC.As the pKa of GEM is 3.6, the pH of its solution is 2.7-3.6 (3.6 in this study), thus causing a concern of chemical cystitis [25,27].However, in the current preliminary study, the symptom of chemical cystitis was not observed and will be further researched in the subsequent main trial.
All patients enrolled in this study had HRNMIBC, including two with CIS.In this cohort, one-year cumulative incidence of recurrence was 23.8% for EORTC intermediate risk (recurrence score 5-9) group and 37.5% for high risk (recurrence score 10-17) group, while the predicted probability of recurrence was 38% (95% CI: 35%,41%) and 61% (95% CI: 55%,67%), respectively [2].It was noteworthy that one of two patients with primary CIS remained recurrence free during the 18-month follow-up.However, tumor recurrence was detected in the other patient during his three-month cystoscopy, causing confusion as to whether it was recurrent or remnant.Fortunately, in our study, no serious complication occurred, and no patient had a GEM allergy.AEs of acute cystitis and pain were considered to be linked with the indwelling catheter, and all patients who experienced AEs recovered completely.Previously published clinical trials on different hyperthermia technologies documented urinary tract infection and sepsis in 0-23% of patients, strictures in 0-10%, allergic reactions in 0-15%, hematuria in 2-62%, and grade 3 or higher AEs in 10-12% of the patients [5,19,28].With similar reports of AEs in our study, the new regimen of BR-PRG with GEM can be considered to be safe and well tolerated.
Despite its merits, this trial had a few limitations.The presented results were derived from a small-scale pilot study and lacked a control group; however, the trial fulfilled its primary objective of evaluating preliminary safety of the proposed novel HIVEC device with GEM as the chemotherapeutic agent.A pilot study itself is exploratory and tries to answer questions such as the pragmatics of recruitment and whether a larger trial is feasible.We tried to investigate and answer questions regarding the stability of intravesical GEM during HIVEC, as well as the heating efficiency and safety of the novel device, BR-PRG.However, the absorption rate, effects of dilution, viscosity, and plasma concentrations were not tested in this study, while hematologic parameters in routine blood examination revealed no concerning trends suggesting bone marrow suppression.Finally, the treatment protocol comprised of six to eight weekly HIVEC administrations followed by ten monthly intravesical instillations of GEM alone, which could jeopardize further oncologic outcomes.Hence, an additional large scale study with adequate regime of HIVEC is currently being conducted at our center to evaluate the long-term safety and oncologic potential of BR-PRG with GEM.

Conclusion
GEM has good thermal stability, making it an ideal drug for use in HIVEC.The novel device BR-PRG proved to be safe and effective in delivering HIVEC, in combination with GEM, demonstrating some superiority over the existing systems.
The long-term safety and oncological potential of BR-PRG with GEM need to be evaluated further.

Figure 1 .
Figure 1.(A) Design of the BR-PRG type HIVEC device.‹ Metal drug container ›Heating system fi Vent valve fl Dose valve °Peristaltic pump -Short circuit loop † Outlet pipe ‡ Inlet pipe.(B) Schematic diagram of the pipeline system.

Figure 3 .
Figure 3. (A) External picture of the BR-PRG device.(B) Topological graph of the BR-PRG.

Figure 4 .
Figure 4.The content percentage (%) of GEM at various temperatures compared to 25 C.

Figure 5 .
Figure 5. Cumulative incidence of recurrence for A) Intermediate risk and B) High risk patients.

Table 1 .
Patient and tumor characteristics of 18 HRNMIBC patients.