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Boron neutron capture therapy is a promising binary treatment for cancer. It is based on the nuclear fission that occurs when non-radioactive 10B absorbs thermal neutrons. One of the two boron compounds currently used in clinical trials for this therapy is BSH. To ensure differentiated retention in the tumour versus normal tissue prior to treatment, routine analytical methods to determine pharmacokinetics must be available. For this purpose we have developed a new, easy and time saving approach, in which the separation of boron derivatives is performed by means of capillary electrophoresis (CE). The CE method allows analyses to be performed in short times (less than 18 min), sensitively (LOD 8 pg loaded on the capillary) quantitatively (LOQ 5 microg/ml) and with a high efficiency of separation. Moreover it is simpler than HPLC and more reproducible (intra- and inter-day values were +/-1% and +/-3%, respectively), and does not require a specific column of derivatization. Mass spectrometry analysis of boron derivatives in different samples was also performed to ensure correct attribution of the CE peaks.

Antiplasmodial activities versus the chloroquine sensitive D10 strain of Plasmodium falciparum of a series of N(1),N(1)-diethyl-N(2)-(4-quinolinyl)-1,2-ethanediamines with 11 different substituents at the 7-position on the quinoline ring have been investigated in vitro. Electron-withdrawing groups at the 7-position have been shown to lower the pK(a) of both the quinoline ring nitrogen atom and the tertiary amino nitrogen in the alkyl side chain. The quinoline nitrogen pK(a) ranges from 6.28 in the nitro derivative to 8.36 in the amino derivative, while the tertiary amino nitrogen has a pK(a) ranging between 7.65 in the trifluoromethyl derivative and 10.02 in the amino derivative. Calculation suggests that the resulting pH trapping of these compounds in the parasite food vacuole ranges between about 7% of that observed in chloroquine for the NO(2) derivative and 97% in the amino derivative. A direct proportionality between antiplasmodial activity normalized for pH trapping and beta-hematin inhibitory activity was observed. Activity could not be correlated with any other observed physical parameter. The beta-hematin inhibitory activity of these derivatives appears to correlate with both the hematin-quinoline association constant and the electron-withdrawing capacity of the group at the 7-position (Hammett constant). For the compounds under investigation, the hematin association constant is in turn influenced by the lipophilicity of the group at the 7-position.

Intraerythrocytic plasmodia form hemozoin as a detoxification product of hemoglobin-derived heme. An identical substance, beta-hematin (BH), can be obtained in vitro from hematin at acidic pH. Quinoline-antimalarials inhibit BH formation. Standardization of test conditions is essential for studying the interaction of compounds with this process and screening potential inhibitors. A spectrophotometric microassay of heme polymerization inhibitory activity (HPIA) (Basilico et al., Journal of Antimicrobial Chemotherapy 42, 55-60, 1998) previously reported was used to investigate the effect of pH and salt concentration on BH formation. The yield of BH formation decreased with pH. Moreover, under conditions used in the above HPIA assay (18 h, 37 degrees C, pH = 2.7), several salts including chloride and phosphate inhibited the process. Aminoquinoline drugs formulated as salts (chloroquine-phosphate, primaquine-diphosphate), but not chloroquine-base, also inhibited the reaction. Interference by salts was highest at low pH and decreased at higher pH (pH 4). Here, we describe different assay conditions that eliminate these problems (BHIA, beta-hematin inhibitory activity). By replacing hematin with hemin as the porphyrin and NaOH solution with DMSO as solvent, the formation of BH was independent of pH up to pH 5.1. No interference by salts was observed over the pH range 2.7-5.1. Dose-dependent inhibition of BH formation was obtained with chloroquine-base, chloroquine-phosphate, and chloroquine-sulfate at pH 5.1. Primaquine was not inhibitory. The final product, characterized by solubility in DMSO, consists of pure BH by FT-IR spectroscopy. The BHIA assay (hemin in DMSO, acetate buffer pH 5 +/- 0.1, 18 h at 37 degrees C) is designed to screen for those molecules forming pi-pi interactions with hematin and thus inhibiting beta-hematin formation.

To assess whether molecular oxygen and oxidative stress contribute to chloroquine activity, we cultivated strains of Plasmodium falciparum in erythrocytes with carboxyhemoglobin and an atmosphere containing 2% CO, 5% CO 2 and 93% N 2 . Results indicate that, contrary to common belief, oxygen is not involved in the activity of chloroquine. Reactive radicals formation is suggested.