Formulation and bioavilability study of a gemcitabine intravesical thermoreversible gel for bladder carcinoma treatment

Background and aims
We have been requested to develop a intravesical formulation of thermoreversible gemcitabine gel 28.5 mg/mL, (GTG). Herein, we present the three steps for the drug development: the setting up of a quantitative method for the determination of gemcitabine and its degradation product, the rheological formulation study and an ex vivo bioavailability.

Materials and methods
GTG was mixed with (5:1) P407/P188 poloxamers at 28.5 mg/mL while the reference medicine of gemcitabine (SPG) was 38 mg/mL. The analysis was conducted with a high performance liquid chromatography (HPLC) coupled with a double detection: ultraviolet with a diode array detector (DAD) and mass spectrometry with a simple quadrupole (SQ). The HPLC system consisted of a C18 stationary phase (150 mm x 2.1 mm, 2.6 µm) thermostated at 20 °C and a mobile phase of ammonium formate buffer (pH = 3) / methanol + 2% formate buffer in gradient mode with a 0.4 mL/min flow rate. The SQ was composed by an electrospray ionization source with N2 gas temperature at 350 °C and flow rate and 12 L/min. The capillary voltage was 3 kV. The m/z transition of the gemcitabine and its internal standard ([13C, 15N2] gemcitabine hydrochloride) were respectively 264 and 267. The m/z transition of the degradation products were 112, 265, 205 and 336 for the impurities A (cytosine), C (1-(2-deoxy-2,2-difluoro-β-D-erythro-pentofuranosyl)pyrimidin-2,4(1H,3H)-dione(2′-deoxy-2′,2′-difluorouridine) specified in the Eur.Ph, PD1 and PD2 found. Finally, a DAD spectrum was performed between 200 and 400 nm to ensure the absence of any other non-specified impurities. The validation procedure was fulfilled accordingly to the GERPAC and SFSTP guidelines. The rheological formulation and ex vivo bioavailability study consisted of an instillation by endo-vesical probe of GTG and SPG on 2 porcine bladders maintained at 37 °C and treated for 2 h. At the end of the 2 h, the injected volumes of GTG and SPG were removed and rinsed with 50 mL of 0.9% NaCl. Three urothelium samples were taken randomly from each bladder and ground. The wash solution as well as the grounded samples were analyzed by HPLC-DAD-SQ.

Results
The method validation criterions were met linearity R² = 0.997 (R² > 0.99), repeatability (1.96% < 5 %), reproducibility (2.48% < 8 %), trueness (0.69% < 10%). The tolerance interval bounds of the accuracy profiles were within the limits of acceptability. The GTG formulation resulted in increased significantly gemcitabine concentration in the rinsing solution 20.27 ± 0.47 mg/g and in the urothelium 54.42 ± 1.95 mg/g compared to 12.09 ± 0.15 mg/g and 40.25 ± 1.11 mg/g with the GSP.

Discussion/ Conclusion
The determination method stability indicator in the different matrices of interest (poloxamers and epithelium) was validated. The use of a thermoreversible gel formulation was effective in improving the bioavailability of gemcitabine into urothelium. The efficiency of this new formulation will be evaluated in a clinical trial.