Synthesis and in vivo murine evaluation of Na4[1-(1'-B10H9)-6-SHB10H8] as a potential agent for boron neutron capture therapy.

Reaction of the normal isomer of [B20H18]2- and the protected thiol anion, [SC(O)OC(CH3)3]-, produces an unexpected isomer of [B20H17SC(O)OC(CH3)3]4- directly and in good yield. The isomer produced under mild conditions is characterized by an apical-apical boron atom intercage connection as well as the location of the thiol substituent on an equatorial belt adjacent to the terminal boron apex. Although the formation of this isomer from nucleophilic attack of the normal isomer of [B20H18]2- has not been reported previously, the isomeric assignment has been unambiguously confirmed by one-dimensional and two-dimensional 11B NMR spectroscopy. Deprotection of the thiol substituent under acidic conditions produces a protonated intermediate, [B20H18SH]3-, which can be deprotonated with a suitable base to yield the desired product, [B20H17SH]4-. The sodium salt of the resulting [B20H17SH]4- ion has been encapsulated in small, unilamellar liposomes, which are capable of delivering their contents selectively to tumors in vivo, and investigated as a potential agent for boron neutron capture therapy. The biodistribution of boron was determined after intravenous injection of the liposomal suspension into BALB/c mice bearing EMT6 mammary adenocarcinoma. At low injected doses, the tumor boron concentration increased throughout the time-course experiment, resulting in a maximum observed boron concentration of 46.7 micrograms of B per g of tumor at 48 h and a tumor to blood boron ratio of 7.7. The boron concentration obtained in the tumor corresponds to 22.2% injected dose (i.d.) per g of tissue, a value analogous to the most promising polyhedral borane anions investigated for liposomal delivery and subsequent application in boron neutron capture therapy.

Selective boron delivery to murine tumors by lipophilic species incorporated in the membranes of unilamellar liposomes.

The nido-carborane species K[nido-7-CH3(CH2)15-7,8-C2B9H11] has been synthesized for use as an addend for the bilayer membrane of liposomes. Small unilamellar vesicles, composed of distearoylphosphatidylcholine/cholesterol, 1:1, and incorporating K[nido-7-CH3(CH2)15-7,8-C2B9H11] in the bilayer, have been investigated in vivo. The time-course biodistribution of boron delivered by these liposomes was determined by inductively coupled plasma-atomic emission spectroscopy analyses after the injection of liposomal suspensions in BALB/c mice bearing EMT6 mammary adenocarcinomas. At the low injected doses normally used (approximately 5-10 mg of boron per kg of body weight), peak tumor boron concentrations of approximately 35 micrograms of boron per g of tissue and tumor/blood boron ratios of approximately 8 were achieved. These values are sufficiently high for the successful application of boron neutron capture therapy. The bilayer-embedded boron compound may provide the sole boron source or, alternatively, a concentrated aqueous solution of a hydrophilic boron compound may also be encapsulated within the liposomes to provide a dose enhancement. Thus, the incorporation of both K[nido-7-CH3(CH2)15-7,8-C2B9H11] and the hydrophilic species, Na3[1-(2'-B10H9)-2-NH3B10H8], within the same liposomes demonstrated significantly enhanced biodistribution characteristics, exemplified by maximum tumor boron concentrations of approximately 50 micrograms of boron per g of tissue and tumor/blood boron ratios of approximately 6.

Na3[B20H17NH3]: synthesis and liposomal delivery to murine tumors.

The polyhedral borane ion [n-B20H18]2- reacts with liquid ammonia in the presence of a suitable base to produce an apical-equatorial (ae) isomer of the [B20H17NH3]3- ion, [1-(2'-B10H9)-2-NH3B10H8]3-. The structure of this product has been confirmed by 11B NMR spectroscopy and x-ray crystallography. This species undergoes acid-catalyzed rearrangement to an apical-apical (a2) isomer, [1-(1'-B10H9)-2-NH3B10H8]3-, whose structure has been determined by 11B NMR spectroscopy. The sodium salts of both the ae and the a2 isomers of [B20H17NH3]3- have been encapsulated within small unilamellar liposomes, composed of distearoyl phosphatidylcholine/cholesterol (1:1), and investigated as boron-delivery agents for boron neutron capture therapy (BNCT) of cancer. The biodistribution of boron was determined after the injection of liposomal suspensions into BALB/c mice bearing EMT6 tumors. Both [B20H17NH3]3- isomers exhibited excellent tumor uptake and selectivity at very low injected doses, achieving peak tumor boron concentrations of 30-40 micrograms of B/g of tissue and tumor/blood boron ratios of approximately 5. The enhanced retention of the [B20H17NH3]3- isomers by EMT6 tumors may be attributed to their facile intracellular oxidation to an extremely reactive NH3-substituted [n-B20H18]2- ion, the electrophilic [B20H17NH3]- ion. Both isomers of [B20H17NH3]3- are at least 0.5 V more easily oxidized than other previously investigated species containing 20 boron atoms. In another experiment, [ae-B20H17NH3]3- was encapsulated in liposomes prepared with 5% PEG-2000-distearoyl phosphatidylethanolamine in the liposome membrane. As expected, these liposomes exhibited a longer circulation lifetime in the biodistribution experiment, resulting in the continued accumulation of boron in the tumor over the entire 48-hr experiment and reaching a maximum of 47 micrograms of B/g of tumor.

Model studies directed toward the boron neutron-capture therapy of cancer: boron delivery to murine tumors with liposomes.

The successful treatment of cancer by boron neutron-capture therapy (BNCT) requires the selective concentration of boron-10 within malignant tumors. The potential of liposomes to deliver boron-rich compounds to tumors has been assessed by the examination of the biodistribution of boron delivered by liposomes in tumor-bearing mice. Small unilamellar vesicles with mean diameters of 70 nm or less, composed of a pure synthetic phospholipid (distearoyl phosphatidylcholine) and cholesterol, have been found to stably encapsulate high concentrations of water-soluble ionic boron compounds. The hydrolytically stable borane anions B10H10(2-), B12H11SH2-, B20H17OH4-, B20H19(3-), and the normal form and photoisomer of B20H18(2-) were encapsulated in liposomes as their soluble sodium salts. The tissue concentration of boron in tumor-bearing mice was measured at several time points over 48 h after i.v. injection of emulsions of liposomes containing the borane anions. Although the boron compounds used do not exhibit an affinity for tumors and are normally rapidly cleared from the body, liposomes were observed to selectively deliver the borane anions to tumors. The highest tumor concentrations achieved reached the therapeutic range (greater than 15 micrograms of boron per g of tumor) while maintaining high tumor-boron/blood-boron ratios (greater than 3). The most favorable results were obtained with the two isomers of B20H18(2-). These boron compounds have the capability to react with intracellular components after they have been deposited within tumor cells by the liposome, thereby preventing the borane ion from being released into blood.