Reduced Toxicity of Liposomal Nitrogen Mustard Prodrug Formulation Activated by an Intracellular ROS Feedback Mechanism in Hematological Neoplasm Models.

Nitrogen mustard (NM) is among the earliest drugs used to treat malignant tumors and it kills tumor cells by cross-linking DNA. Unfortunately, because of the short half-life and unfavorable selectivity, NM causes significant damage to normal tissues. As NM can increase the levels of reactive oxygen species (ROS) in tumor cells, a ROS-activated nitrogen mustard prodrug (NM-Pro) was synthesized and mixed with NM at a specific ratio to obtain an "NM-ROS-NM-Pro-NM" positive feedback system, which ultimately achieves a specific lethal effect on hematological neoplasms. The further encapsulation of NM/NM-Pro in liposomes allows the sustained release of the drug and prolongs the residence time in vivo. Here, we prepared stable liposomes with a uniform particle size of 170.6 ± 2.2 nm. The optimal ratio of NM to NM-Pro in this study was 2:1. The active drug NM in the NM/NM-Pro system continuously stimulated ROS production by the cells, which in turn further activated the NM-Pro to continuously generate NM. The positive feedback pathway between the NM and NM-Pro resulted in the specific death of tumor cells. Furthermore, the K562 hematological neoplasm model was utilized to evaluate the therapeutic effect of NM/NM-Pro liposomes in vivo. After encapsulation in liposomes, the targeting of tumor cells was increased approximately two times compared with that of normal cells, and NM/NM-Pro liposomes exhibited reduced toxicity, without an increase in drug activity compared to the NM/NM-Pro combination. The NM/NM-Pro delivery system exerts a positive feedback effect on ROS production in tumor cells and displays good potential for the specific killing of hematoma cells.

Mechanistic study of IR-780 dye as a potential tumor targeting and drug delivery agent.

IR-780 iodide, a near-infrared fluorescent heptamethine dye, has been recently characterized to exhibit preferential accumulation property in the mitochondria of tumor cells. In this study, we investigated the possible mechanisms for its tumor selective activity and its potential as a drug delivery carrier. Results showed that the energy-dependent uptake of IR-780 iodide into the mitochondria of tumor cells was affected by glycolysis and plasma membrane potential. Moreover, OATP1B3 subtype of organic anion transporter peptides (OATPs) may play a dominant role in the transportation of IR-780 iodide into tumor cells, while cellular endocytosis, mitochondrial membrane potential and the ATP-binding cassette transporters did not show significant influence to its accumulation. We further evaluated the potential of IR-780 iodide as a drug delivery carrier by covalent conjugation of IR-780 with nitrogen mustard (IR-780NM). In vivo imaging showed that IR-780NM remained the tumor targeting property, indicating that IR-780 iodide could be potentially applied as a drug delivery agent for cancer targeted imaging and therapy.

Quantitation of biomarkers of exposure to nitrogen mustards in urine from rats dosed with nitrogen mustards and from an unexposed human population.

The nitrogen mustards bis(2-chloroethyl)ethylamine (HN1), bis(2-chloroethyl)methylamine (HN2), and tris(2-chloroethyl)amine (HN3) have the potential to be used as chemical terrorism agents because of their extreme vesicant properties. We modified a previously reported method to incorporate automated solid-phase extraction, improve chromatography, and include the urinary metabolite for HN3. The improved method was used to measure levels of the urinary metabolites N-ethyldiethanolamine (EDEA), N-methyldiethanolamine (MDEA), and triethanolamine (TEA) in rats dosed with HN1, HN2, and HN3, respectively, and to establish background levels of EDEA, MDEA, and TEA in human urine samples from a population with no known exposure to nitrogen mustards. Rat dosing experiments confirmed that EDEA, MDEA, and TEA could be detected in urine for at least 48 h after exposure to HN1, HN2, and HN3, respectively. Substantial amounts of EDEA (89 ng/mL), MDEA (170 ng/mL), and TEA (1105 ng/mL) were measured in the urine of rats exposed to 10 mg HN1, HN2, and HN3, respectively, 48 h after exposure. The background concentrations for TEA in the human population ranged from below the limit of detection (LOD 3 ng/mL) to approximately 6500 ng/mL. Neither EDEA (LOD 0.4 ng/mL) nor MDEA (LOD 0.8 ng/mL) was detected above the LOD in the human samples.

Covalent sequestration of the nitrogen mustard mechlorethamine by metallothionein.

The research reported here demonstrates covalent binding to the metal-binding protein metallothionein (MT) by the therapeutic nitrogen mustard mechlorethamine. The most surprising aspect of this interaction is the selectivity of the alkylating agent for specific residues of MT. A combination of MS and proteolytic and enzymatic methods was used to deduce specific locations of mechlorethamine alkylation. These experiments indicated that alkylation occurs predominantly in the carboxyl domain of MT, with one molecule of mechlorethamine covalently cross-linking two cysteine residues. Electrospray MS revealed the retention of all seven metal ions in the cross-linked MT/mechlorethamine adducts, highlighting the uniqueness of this protein. Computerized docking experiments supported the hypothesis that selective binding precedes selective alkylation, and the structure of the drug indicates the minimal structural requirements for this binding. These results support the idea that MT overexpressed in tumor cells contributes to the inactivation of anticancer drugs.

Innovative treatment approaches for rheumatoid arthritis. Cyclosporin, leflunomide and nitrogen mustard.

Cyclosporin A (CSA) Cyclosporin inhibits IL-2 release and T-cell activation and, secondarily, affects B-cell function. It also inhibits bone resorption, at least in vitro. This drug's bio-availability averages 25-35% but is highly variable. Food and grapefruit juice enhance bio-availability and newer formulations may make its absorption more reliable. It is highly concentrated in fatty tissues and red blood cells but does not cross the blood-brain barrier. CSA is metabolized to numerous metabolites by the liver and its elimination half-life is 6-12 hours in the absence of severe liver disease. Biliary excretion accounts for 94% of CSAs elimination. Because it is highly metabolized, its metabolism can be inhibited by other drugs (e.g. ketoconazole and erythromycin) or its metabolism can be induced (e.g. anticonvulsants). Cyclosporin is more effective than placebo for the treatment of rheumatoid arthritis and as effective as other antirheumatics. There is potential for the use of CSAs in DMARD combinations. The principal toxicities of cyclosporin are gastro-intestinal and renal, with the latter being of more concern. Leflunomide (LF). Leflunomide may be a pyrimidine synthesis inhibitor, although tyrosine kinase inhibitor may also be part of its mechanism of action. Its active metabolite is excreted renally to a large degree, with a prolonged elimination half-life of about 11 days. Since LF is activated by liver metabolism, renal failure may have less effect on kinetics than severe liver disease. Early data on efficacy indicate efficacy at 10-25 mg/day, although more well-controlled data is necessary. Toxicity relates to the skin, liver and GI tract, although some degree of weight loss was also found. Nitrogen mustard (NM). Nitrogen mustard is an alkylating agent whose pharmacokinetics are poorly understood. Small, open studies in RA indicate that NM has a potential for relatively rapid response (1-2 weeks) but, clearly, much work remains to be done. As an alkylating agent, GI and hematological toxicities are of greatest concern.

Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.

Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze the conjugation, and thus, the detoxication of a structurally diverse group of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards. We proposed that structural alteration of the nonspecific electrophile-binding site would produce mutant enzymes with increased efficiency for detoxication of a single drug and that these mutants could serve as useful somatic transgenes to protect healthy human cells against single alkylating agents used in cancer chemotherapy protocols. Random mutagenesis of three regions (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selection of Escherichia coli expressing the enzyme library with the nitrogen mustard mechlorethamine (20-500 microM), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechlorethamine conjugation (up to 15-fold increase in kcat and up to 6-fold increase in kcat/Km) and that confer up to 31-fold resistance, which is 9-fold greater drug resistance than that conferred by the wild-type enzyme. The results suggest a general strategy for modification of drug- and carcinogen-metabolizing enzymes to achieve desired resistance in both prokaryotic and eukaryotic plant and animal cells.

Genotoxic effects of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) and nitrogen mustard-N-oxide (nitromin) in Walker carcinoma cells under aerobic and hypoxic conditions.

As judged by alkaline elution, exposure of Walker cells to either 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) or nitromin results in a dose-dependent increase in DNA damage due to single-strand breaks. With nitromin or SR 4233 there was little difference in the extent of DNA single-strand breaks between Walker cells incubated either hypoxically or aerobically. In contrast, there was a 24-fold enhancement in the differential hypoxic/aerobic response to SR 4233 in clonogenic studies. Following incubation of cells with nitrogen mustard, DNA cross-linking is observed. Bioreduction of nitromin would be expected to yield nitrogen mustard as the putative reactive metabolite. However, only DNA strand-breaks could be detected in Walker cells incubated with nitromin, suggesting that reduction of this pro-drug to nitrogen mustard was not a major activation pathway. In cells incubated under aerobic conditions, SR 4233 induces oxidative DNA damage, as indicated by the formation of 8-hydroxydeoxyguanosine, suggesting the involvement of futile redox cycling. In rats dosed with SR 4233 in vivo, significantly higher levels of 8-hydroxydeoxyguanosine could be detected in liver, compared to vehicle-dosed controls.

Acute lesions in rats caused by 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) or nitromin: a comparison with rates of reduction in microsomal systems from target organs.

Pathological lesions to male Fischer rats were investigated 24 h after the administration of 3-amino-1,2,4-benzotriazine-1,4- dioxide (SR 4233) or nitromin, two compounds which need to undergo bioreductive activation in order to exert their toxic effects. Although SR 4233 reduction leads to a putative free radical species while with nitromin a bifunctional alkylating agent is formed, in both instances, the bone marrow was a major target organ. However, the response of other organs to these compounds differed. SR 4233 caused lesions to the olfactory epithelium, liver, kidney and thymus. Nitromin caused focal haemorrhages on the intestine, which were reduced in germ-free rats. Rates of reduction of SR 4233 or nitromin were determined under anaerobic conditions using microsomal preparations from target tissues. With SR 4233 as a substrate, reductase activities were highest in the olfactory epithelium, 6 fold higher than in the liver. SR 4233 reductase activities generally correlated with those of NADPH:cytochrome c reductase or the concentration of cytochrome P-450 reductase protein in the affected organs while with nitromin, there appeared to be no such relationship. The present results support the concept that the expression of pathological damage in vivo is a multifactorial process and does not directly correlate with initial rates of reduction of either drug determined in vitro.

[14C]mechlorethamine binding to proteins of the human keratinocyte.

Much mustard agent research has focused on mustard/DNA interactions. Mustard also interacts with proteins, however, and to reach the DNA any agent must first pass through the cytoplasm. We hypothesized that the cell's proteins would covalently bind mustard, and thereby limit its access to the DNA. Keratinocyte proteins were radiolabeled with [14C]mechlorethamine and separated by electrophoresis. The banding patterns that resulted were made visible on x-ray films, then compared with control patterns. A correspondence of almost one-to-one was observed, which supports the hypothesis that many cellular proteins are susceptible to mustard alkylation. It follows that some mustard symptoms probably result from effects on existing proteins.

In vitro inhibition of nitrogen mustard efficacy by postincubation of treated cells in serum protein.

Lettre-Ehrlich cells were loaded with sufficient HN2 to produce about a 98% cell kill. Postincubation of the HN2-loaded cells in PBS resulted in the loss of about 40% of their HN2 without changing the cytolytic effect, supporting the proposal that only bound drug was effective. Postincubation of the HN2-loaded cells in PBS which contained 2% bovine serum albumin or in cell-free mouse ascitic fluid (1.8% protein) resulted in the same relative cellular HN2 loss as well as a 79% decrease in the cell kill. The cytolytic effect of HN2 is believed to be dependent on the degree to which the drug crosslinks DNA in 2 sequential reactions. It seems likely that such crosslinking occurred in nearly all of the PBS-postincubated cells, as they were nearly all killed. By analogy, albumin postincubation apparently blocked the competition of such crosslinking.

Choline uptake is increased by Ca++ and liposomes+ Ca++ in ascites tumor cells.

Steady-state uptake of choline by Lettre-Ehrlich tumor cells in vitro, resulting in cell-to-medium ratios of 10 or more, is significantly increased by 0.2-1.0 mM Ca++ as well as by dipalmitoyl phosphatidyl choline multilamellar liposomes + Ca++. The increases occur in spite of a decrease in carrier affinity, as indicated by the Km, and therefore result either from increased carrier velocity or utilization of new carriers. About half of the labelled choline which is taken up is firmly bound to cells. That label which freely leaves cells is phosphocholine, thus, these cells utilize choline mainly in phospholipid synthesis. Choline and nitrogen mustard (HN2) share a plasma membrane carrier but the intracellular distribution of HN2 into DNA, RNA and protein, contrasts with that of choline, into phospholipid.