Effect of particle size of nanopaticulate L-BPA formulation on biodistribution of 10 B after its intratumoral administration to tumor-bearing mice KOBE GAKUIN UNIVERSITY 1 Laboratory of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences and Cooperative Research Center of Life Sciences, Kobe Gakuin University, Kobe , Japan. 2 Department of Orthopaedic Surgery, Hyogo Cancer Center, Akashi , Japan. T. Andoh 1, T. Fujimoto 2, Y. Fukumori 1 and H. Ichikawa 1. 1 2 Take home message KOBE GAKUIN UNIVERSITY Intratumoral administration of nano-sized BPA suspension would show a potential ability to be used as a BNCT reagent. Background 3 KOBE GAKUIN UNIVERSITY Therefore, a more effective 10 B carrier is required for making BNCT more successful. While L-BPA accumulates into tumor cells spontaneously, it has some drawbacks such as poor water-solubility and a rapid decrease of 10 B concentration in tissue after administration. p-borono-L-phenylalanine (L-BPA) has been widely employed as a 10 B agent in the clinical BNCT. The successful treatment of cancer by BNCT requires the selective delivery of large amounts of 10 B to tumor cells. L-BPA Nanosuspension (BPA-NS) for BNCT BPA-NS is nano-sized particle dispersion composed of L-BPA itself. BPA-NS can give a prolonged retain-ability of 10 B in tumor tissue in comparison to BPA-Fr if its intratumoral (i.t.) administration can be applied to certain tumors. After intravenous (i.v.) administration, tumor-selective accumulation can be expected due to the so-called enhanced permeability and retention effect (EPR effect), provided that the BPA-NS can possess a long-circulating property in blood. L-BPA crystals Tumor tissue Normal tissue Blood vessel L-BPA Nanosuspension (BPA-NS) 4 KOBE GAKUIN UNIVERSITY Wet- milling Intravenous administration Intratumoral administration L-BPA The aim of this study is to develop the BPA-NS formulation and to assess its biodistribution followed by i.v. or i.t. administration to tumor-bearing mice. A: Takashima S. et al, AJR, 151, 1988 B: Yamaguchi T. et al, Jpn J Gastroenterol Surg, 19(1), 1986 The formation mechanism of spaces (Cavity or Cyst) in the tumor can be divided into two major types. 1. Cavity formation due to central necrosis caused by inadequate blood supply and by infection as a consequence of tumor growth. (A) 2. Cyst formation due to canceration of the fluid-secreting cell (pancreas, ovary, breast, or gastrointestinal tract). (B) Treatment other than surgical resection is lacking. The cavity or cyst can be an administration site for BPA-NS. A: Cavity-forming lung metastasis melanoma B: Cyst of pancreas KOBE GAKUIN UNIVERSITY Possible target cancers in the application of intratumorally injectable BPA-NS 5 Solid tumor model The culture medium containing 710 6 of B16F10 mouse melanoma cells was subcutaneously (s.c.) implanted into the right hip of 5-weeks-old C57BL/6J mice, until it formed tumor mass with mm in diameter (typically at 7-10 days after implantation). Cavity-forming model The cavity was formed by removing the contents from the solid tumor mass of the mice through a syringe. KOBE GAKUIN UNIVERSITY Preparation of tumor-bearing animal models 6 Solid tumor model Cavity-forming model C57BL/6J mouse KOBE GAKUIN UNIVERSITY Preparation of BPA-NSs Planetary ball-milling (Pulverisette-7) L-BPA Nanosuspension (BPA-NS) L-BPA Nanosuspension (BPA-NS) Wet-milling process (Planetary ball-milling) Agate pot (12-mL volume) 7 L-BPA Solutol HS15 Soybean Lecithin MQ water Zirconia beads 250 mg 93 mg 32 mg 5 mL 30 g Cumulative (%) Particle size (m) Formulation Concentration (mg L-BPA/mL) Mass median diameter (nm) BPA-Fr50.2 NS NS Fig 1. Particle size distribution Table 1. Particle size and L-BPA concentration :NS 200 :NS 400 Time (hour) 10 B concentration ( 10 B g/g wet tissue) Fig B accumulation in tumor and blood, 0.083, 0.5, 1, 3, 6, 12 and 24 hour after i.v. injection (24 mg 10 B/kg b.w.) of BPA-Fr () or BPA-NS200 () to B16F10 bearing mice. Each value represents the mean S.D. (n=3). KOBE GAKUIN UNIVERSITY BPA-Fr () BPA-NS200 () Tumor Blood 8 BPA-Fr and BPA-NS200 (500 mg BPA/kg) were intravenously (i.v.) administered via femoral vein to solid tumor model. At predetermined time intervals after dosing, the mice were sacrificed with diethyl ether. Tissue samples were collected immediately. Retention of 10 B after i.v. administration 30 ppm 49 ppm Tumor Time (hour) 10 B concentration ( 10 B g/g wet tissue) Fig B accumulation in tumor and blood, 0.083, 0.5, 1, 3, 6, 12 and 24 hour after ) i.t. injection (24 mg 10 B/kg b.w.) of BPA-Fr (), BPA-NS200 () or BPA-NS400 () to B16F10 bearing mice. Each value represents the mean S.D. (n=3). KOBE GAKUIN UNIVERSITY BPA-Fr () BPA-NS200 () BPA-NS400 () Blood 9 BPA-Fr, BPA-NS200 and NS400 (500 mg BPA/kg) were intratumorally (i.t.) administered to cavity-forming model. Retention of 10 B after i.t. administration 108 ppm (61%) 284 ppm (80%) 29 ppm (11%) After intravenous administration, short retention time of BPA-NS in blood may account for such a lower boron concentration in tumor. After intratumoral administration, BPA-NSs showed much higher 10 B concentration in tumor than BPA-Fr. The retention time of BPA-NSs in tumor was depended on the particle size of BPA crystals in BPA-NSs. KOBE GAKUIN UNIVERSITY 10 Short summary KOBE GAKUIN UNIVERSITY Diffusion behavior of BPA-NSs in tumor tissue Upper Lower Middle BPA formulations 11 Purpose To Investigate whether BPA-NSs can diffuse well into tumor tissue. Methods BPA-Fr, BPA-NS200 and -NS400 (250 mg BPA/kg in dose) were intratumorally administered to the interfacial site of solid tumor model. At a predetermined time after administration, the mice were sacrificed, and then tumor samples were divided into three sections (upper, middle and lower sections). KOBE GAKUIN UNIVERSITY Fig B concentration-time profiles in tumor tissues divided into three sections (upper, middle and, lower) after intratumoral injection of BPA-Fr to melanoma-bearing mice Dose: BPA-NS, 12 mg 10 B/kg. Each value represents the mean S.D. (n=3). 10 B concentration ( 10 B g/g tumor) Upper Middle Lower Whole tumor 6% 36% 18% 36% 46% 27% 36% 37% 58% min1 hr3 hr 12 Diffusion behavior of BPA-Fr in tumor tissue UMLWhole UML UML KOBE GAKUIN UNIVERSITY Fig B concentration-time profiles in tumor tissues divided into three sections (upper, middle and, lower) after intratumoral injection of BPA-NS200 to melanoma-bearing mice Dose: BPA-NS, 12 mg 10 B/kg. Each value represents the mean S.D. (n=3). 10 B concentration ( 10 B g/g tumor) 7% 18% 75% 12% 38% 50% 28% 33% 39% 5 min1 hr3 hr Diffusion behavior of NS200 in tumor tissue Upper Middle Lower Whole tumor UMLWhole UML UML KOBE GAKUIN UNIVERSITY Fig B concentration-time profiles in tumor tissues divided into three sections (upper, middle and, lower) after intratumoral injection of BPA-NS400 to melanoma-bearing mice Dose: BPA-NS, 12 mg 10 B/kg. Each value represents the mean S.D. (n=3). 10 B concentration ( 10 B g/g tumor) 5 min1 hr3 hr 5% 26% 69% 6% 26% 68% 45% 11% 44% Diffusion behavior of NS400 in tumor tissue Upper Middle Lower Whole tumor UMLWhole UML UML KOBE GAKUIN UNIVERSITY 15 BPA-NS-200 Intratumoral administration of BPA-NSs BPA-NS-400 Tumoral cells Diffusion behavior of BPA-NSs in tumor tissue Diffusion behaviors of BPA-NS200 and BPA-NS400 were influenced by the number/size of particles, the extent of leaching to blood vessel and the rate of dissolution. Favorable diffusion behavior of 10 B in tumor was achievable even in the administration of solid BPA-nanoparticles with appropriate particle size. BPA-NSs were effective to prolong the retention time of 10 B in the tumor tissue with the siginificantly higher concentration, in comparison with BPA-Fr. The particle size of BPA crystals in BPA-NSs was found to be an important factor affecting the diffusion behaviors in tumor; smaller particles can show a good diffusibility. KOBE GAKUIN UNIVERSITY 16 Summary KOBE GAKUIN UNIVERSITY Acknowledgements A part of this work was supported by MEXT- Supported Program for the Strategic Research Foundation at Private Universities, Intratumoral administration of BPA-NSs would show a potential ability to be used as a BNCT reagent. Conclusion 17