Mustard Analog Research Articles

Mass Spectral Behavior of the Hydrolysis Products of Sesqui- and Oxy-Mustard Type Chemical Warfare Agents in Atmospheric Pressure Chemical Ionization

Bis(2-hydroxyethylthio)alkanes and bis(2-hydroxyethylthioalkyl)ethers are important biological and environmental degradation products of sulfur mustard analogs known as sesqui- and oxy-mustards. We used atmospheric pressure chemical ionization mass spectrometry (APCI MS) to acquire characteristic spectra of these compounds in positive and negative ionization modes. Positive APCI mass spectra exhibited [M + H](+); negative APCI MS generated [M + O(2)](-), [M - H](-), and [M - 3H](-); and both positive and negative APCI mass spectra contained fragment ions due to in-source collision-induced dissociation. Product ion scans confirmed the origin of fragment ions observed in single-stage MS. Although the spectra of these compounds were very similar, positive and negative APCI mass spectra of the oxy-mustard hydrolysis product, bis(2-hydroxyethylthiomethyl)ether, differed from the spectra of the other compounds in a manner that suggested a rearrangement to the sesqui-mustard hydrolysis product, bis(2-hydroxyethylthio)methane. We evaluated the [M + O(2)](-) adduct ion for quantification via liquid chromatography-MS/MS in the multiple-reaction monitoring (MRM) mode by constructing calibration curves from three precursor/product ion transitions for all the analytes. Analytical figures of merit generated from the calibration curves indicated the stability and suitability of these transitions for quantification at concentrations in the low ng/mL range. Thus, we are the first to propose a quantitative method predicated on the measurement of product ions generated from the superoxide adduct anion of the sesqui-and oxy-mustard hydrolysis products.

Aneugenic potential of the nitrogen mustard analogues melphalan, chlorambucil and p- N, N-bis(2-chloroethyl)aminophenylacetic acid in cell cultures in vitro

Melphalan (MEL), chlorambucil (CAB) and p-N, N-bis(2-chloroethyl)aminophenylacetic acid (PHE) are nitrogen mustard analogues, which are clinically used as chemotherapeutic agents. They also exert carcinogenic activity. The aim of this study was to investigate the aneugenic potential of the above drugs and the possible mechanism responsible for this activity. The Cytokinesis Block Micronucleus (CBMN) assay in combination with fluorescence in situ hybridization (FISH) was used in human lymphocyte cultures to evaluate micronucleus (MN) frequency. Pancentromeric probe (α-satellite) was applied to identify chromosomes in micronuclei and an X-chromosome specific centromeric probe was used to asses micronucleation and non-disjunction of this chromosome in binucleated cells. The effect of the above compounds on the organization of mitotic apparatus, as a possible target of chemicals with aneugenic potential, was investigated in C 2C 12 mouse cell line by double immunofluorescence of α- and γ-tubulin. We found that the studied drugs increased MN frequency in a linear dose-dependent manner primarily by chromosome breakage and in a lesser extent by an aneugenic mechanism. Non-disjunction and micronucleation of X-chromosome were also induced. Abnormal metaphase cells were linearly increased with concentration and characterized by abnormal centrosome number. Interphase cells with micronuclei and abnormal centrosome number were also observed. Since nitrogen mustards are highly reactive agents, with low selectivity and form covalent bonds with different nucleophilic sites in proteins and nucleic acids, it is reasonable to consider that one possible pathway for nitrogen mustard analogues to exert their aneugenic activity is through reaction with nucleophilic moieties of proteins or genes that are involved in the duplication and/or separation of centrosomes, resulting in abnormal centrosome number. Based on our results the carcinogenicity of nitrogen mustard analogues studied may be attributed not only to their activity to trigger gene mutation and chromosome breakage, but also to their aneugenic potential. Further studies are warranted to clarify the above two hypotheses.

Inhibition of DNA repair in chronic lymphocytic leukemia: Therapeutic implications

16513 Background: Chronic lymphocytic leukemia (CLL) is an indolent leukemia in which there is an accumulation of immature malignant B-lymphocytes. While treatment with chlorambucil, a nitrogen mustard analogue or fludarabine can control the disease, eventually all patients become resistant to chlorambucil/fludarabine. Chlorambucil’s cytotoxicity is mediated by the interstrand crosslinks (ICLs) introduced into DNA. In mammalian cells, two main pathways are known to be involved in double strand break(DSB) repair: homologous recombinational repair (HRR) and nonhomologous endjoining (NHEJ). Methods: Our laboratory has demonstrated that resistance to chlorambucil involved accelerated repair of the ICLs associated with enhanced DNA repair, specifically enhanced nonhomologous endjoining (NHEJ) and homologous recombinational repair (HRR). We also recently demonstrated that gleevec inhibition of c-abl with resulting inhibition of Rad51-related HRR sensitizes CLL lymphocytes in vitro to chlorambucil. This exciting result has stimulated the development of a clinical protocol to test this combination in CLL. Furthermore, our laboratory has demonstrated that wortmannin, a nonspecific inhibitor of DNA-PK (a key component of NHEJ) sensitizes CLL lymphocytes to chlorambucil. Results: We are actually evaluating the effect of NU7026, a relatively specific DNA-PK inhibitor, in the sensitivity of CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and primary CLL-lymphocytes chlorambucil plus NU7026 have a synergistic cytotoxic effect. Noteworthy, the NU7026 doses used in combination with chlorambucil were not toxic to the cells when used alone. Moreover, chlorambucil treatment induced DNA-PK nuclear foci which were inhibited by NU7026 suggesting that the synergy of both drugs is mediated by Nu7026-inhibition of DNA-PK. Conclusion: NU7026 plus chlorambucil should be a useful combination to treat CLL. No significant financial relationships to disclose.

Modulation of the expression of superoxide dismutase gene in lung injury by 2‐chloroethyl ethyl sulfide, a mustard analog

Mustard gas exposure causes inflammatory lung diseases. Many inflammatory lung diseases are associated with oxidative stress. Reactive oxygen species (ROS) are involved in the maintenance of physiological functions. In tissues, it is therefore essential to maintain a steady-state level of antioxidant activity to allow both for the physiological functions of ROS to proceed and at the same time preventing tissue damage. We have recently reported that mustard gas exposure decreases the overall activity of superoxide dismutase (SOD). In the present study, we investigated the effects of mustard gas on each of the three isozymes: SOD-1 (Cu/Zn), SOD-2 (Mn), and SOD-3 (extracellular). Adult guinea pigs were intratracheally injected single doses of 2-chloroethyl ethyl sulfide (CEES) (2 mg/kg body weight) in ethanol. Control animals were injected with vehicle in the same way. The animals were sacrificed after 7 days, and lungs were removed after perfusion with physiological saline. Lung injury was established by measuring the leakage of iodinated-BSA into lung tissue. Mustard gas exposure caused a significant increase in the activity of SOD-1 (35%). However, the SOD-3 activity which is the predominant type in lung was significantly decreased (62%), whereas no change was observed in SOD-2 activity. Thus the decrease in the total activity of SOD was primarily due to the SOD-3 isozyme. Northern blot analysis indicated 3.5-fold increased expression of SOD-1 in mustard gas exposed lung, but no significant change in the expression of SOD-2 and SOD-3 was observed. Mustard gas exposure did not cause mutation in the coding region of SOD-1 gene while causing modulation in expression levels. The protein levels of SOD-1, SOD-2, and SOD-3 were not altered significantly in the mustard gas exposed lung. Our results indicate that the overall decrease in the activity of SOD by mustard gas exposure is probably mediated by direct inactivation of the SOD-3 gene or the enzyme itself. This decrease in the activity of SOD-3 may be due to the cleavage of active form of the protein to an inactive form. The existence of active and inactive forms of SOD-3 as a result of shifts in Cys-Cys disulfide bonding has been described in human, recently. Studies are underway in our laboratory to investigate whether mustard gas induced inactivation of SOD-3 in lung is similarly mediated by a change in Cys-Cys disulfide bonding.

Decrease in brain POMC mRNA expression and onset of obesity in guinea pigs exposed to 2-chloroethyl ethyl sulfide, a mustard analogue

The full spectrum of physiological effects resulting from exposure to sulfur mustard and its analogs is currently unknown. In a guinea pig model, initially selected to study the role of an inflammatory cytokine cascade in mustard gas induced lung injury, we observed significant body weight gain in guinea pigs exposed to an intratracheally injected single dose of 2-chloroethyl ethyl sulfide, a mustard analog. The body weight gain was not associated with any apparent change in appetite. To further elucidate a molecular basis for the observed weight gain, we evaluated candidate genes for the obese phenotype by quantitative RT-PCR. We observed a time- and dose-dependent decrease in guinea pig pro-opiomelanocortin (POMC) message following treatment with mustard gas. This reduction in POMC message is consistent with the onset of obesity in the animals. We hypothesize that the POMC melanocortin pathway provides a mechanistic basis for the observed effects of sulfur mustard on body weight.

Inhibition of cholinephosphotransferase activity in lung injury induced by 2-chloroethyl ethyl sulfide, a mustard analog

Exposure to mustard gas causes inflammatory lung diseases including acute respiratory distress syndrome (ARDS). A defect in the lung surfactant system has been implicated as a cause of ARDS. A major component of lung surfactant is dipalmitoyl phosphatidylcholine (DPPC) and the major pathway for its synthesis is the cytidine diphosphocholine (CDP-choline) pathway. It is not known whether the ARDS induced by mustard gas is mediated by its direct effects on some of the enzymes in the CDP-choline pathway. In the present study we investigated whether mustard gas exposure modulates the activity of cholinephosphotransferase (CPT) the terminal enzyme by CDP-choline pathway. Adult guinea pigs were intratracheally infused with single doses of 2-chloroethyl ethyl sulfide (CEES) (0.5 mg/kg b.wt. in ethanol). Control animals were injected with vehicles only. The animals were sacrificed at different time and the lungs were removed after perfusion with physiological saline. CPT activity increased steadily up to 4 h and then decreased at 6 h and stabilized at 7 days in both mitochondria and microsomes. To determine the dose-dependent effect of CEES on CPT activity we varied the doses of CEES (0.5-6.0 mg/kg b.wt.) and sacrificed the animals at 1 h and 4 h. CPT activity showed a dose-dependent increase of up to 2.0 mg/kg b.wt. of CEES in both mitochondria and microsomes then decreased at 4.0 mg/kg b.wt. For further studies we used a fixed single dose of CEES (2.0 mg/kg b.wt.) and fixed exposure time (7 days). Lung injury was determined by measuring the leakage of iodinated-bovine serum albumin into lung tissue and expressed as the permeability index. CEES exposure (2.0 mg/kg b.wt. for 7 days) caused a significant decrease of both CPT gene expression (approximately 1.7-fold) and activity (approximately 1.5-fold) in the lung. This decrease in CPT activity was not associated with any mutation of the CPT gene. Previously we reported that CEES infusion increased the production of ceramides which are known to modulate PC synthesis. To determine whether ceramides affect microsomal CPT activity the lung microsomal fraction was incubated with different concentrations of C(2)-ceramide prior to CPT assay. CPT activity decreased significantly with increasing dose and time. The present study indicates that CEES causes lung injury and significantly decreases CPT gene expression and activity. This decrease in CPT activity was not associated with any mutation of the CPT gene is probably mediated by accumulation of ceramides. CEES induced ceramide accumulation may thus play an important role in the development of ARDS by modulating CPT enzyme.

Protection by antioxidants against toxicity and apoptosis induced by the sulphur mustard analog 2‐chloroethylethyl sulphide (CEES) in Jurkat T cells and normal human lymphocytes

Protection by antioxidants against toxicity and apoptosis induced by the sulphur mustard analog 2‐chloroethylethyl sulphide (CEES) in Jurkat T cells and normal human lymphocytes

Protection by antioxidants against toxicity and apoptosis induced by the sulphur mustard analog 2-chloroethylethyl sulphide (CEES) in Jurkat T cells and normal human lymphocytes.

1. The mechanism of toxicity of sulphur mustard was investigated by examining the biochemical effects of the analog 2-chloroethylethyl sulphide (CEES) in both human Jurkat cells as well as normal human lymphocytes. 2. Exposure of both types of cells to CEES resulted in a marked decrease in the intracellular concentration of the reduced form of glutathione (GSH), and CEES-induced cell death was potentiated by l-buthionine sulphoximine, an inhibitor of GSH synthesis. 3. CEES increased the endogenous production of reactive oxygen species (ROS) in Jurkat cells, and CEES-induced cell death was potentiated by hydrogen peroxide. 4. CEES induced various hallmarks of apoptosis, including collapse of the mitochondrial membrane potential, proteolytic processing and activation of procaspase-3, and cleavage of poly (ADP-ribose) polymerase. 5. The effects of CEES on the accumulation of ROS, the intracellular concentration of GSH, the mitochondrial membrane potential, and caspase-3 activity were all inhibited by pretreatment of cells with the GSH precursor N-acetyl cysteine or with GSH-ethyl ester. Furthermore, CEES-induced cell death was also prevented by these antioxidants. 6. CEES toxicity appears to be mediated, at least in part, by the generation of ROS and consequent depletion of GSH. Given that sulphur mustard is still a potential biohazard, the protective effects of antioxidants against CEES toxicity demonstrated in Jurkat cells and normal human lymphocytes may provide the basis for the development of a therapeutic strategy to counteract exposure to this chemical weapon.

Evidence of hair loss after subacute exposure to 2-chloroethyl ethyl sulfide, a mustard analog, and beneficial effects of N-acetyl cysteine.

Mustard gas has been used as a vesicant chemical warfare agent. However, a suitable biomarker for monitoring mustard gas exposure is not known. We observed that the hairs of the guinea pigs exposed intratracheally to subacute doses of 2-chloroethyl ethyl sulfide (CEES), a mustard analog, came out very easily though there was no sign of skin lesions or skin damage. Also the hairs looked rough and dry and lost the shiny glaze. There was no recovery from this hair loss, though the animals never became hairless, following CEES exposure. Hairs were observed in this study both visually and with light microscopy. Treatment with N-acetylcysteine (NAC) prior to CEES exposure could prevent the hair loss completely. Hence, sudden hair loss might be a good biomarker for subacute exposure of mustard gas to subjects at risks when the victims might have no other visible symptom of toxicity.

Nitroaryl phosphoramides as novel prodrugs for E. coli nitroreductase activation in enzyme prodrug therapy.

Cyclic and acyclic nitroaryl phosphoramide mustard analogues were activated by E. coli nitroreductase, an enzyme explored in GDEPT. The more active acyclic 4-nitrobenzyl phosphoramide mustard (7) showed 167 500x selective cytotoxicity toward nitroreductase-expressing V79 cells with an IC(50) as low as 0.4 nM. This is about 100x more active and 27x more selective than CB1954 (1). The superior activity was attributed to its better substrate activity (k(cat)/K(m) 19x better than 1) and/or excellent cytotoxicity of phosphoramide mustard released.

Prophylactic protection by N-acetylcysteine against the pulmonary injury induced by 2-chloroethyl ethyl sulfide, a mustard analogue.

Mustard gas exposure causes adult respiratory distress syndrome associated with lung injury. The purpose of this study was to investigate whether an antioxidant, such as N-acetylcysteine (NAC), has any protective effect. Guinea pigs were given single exposure (0.5-6 mg/kg body weight) of 2-chloroethyl ethyl sulfide (CEES) as a mustard analogue intratracheally and maintained for various lengths of time (1 h to 21 days). Within 1 h of CEES infusion at 4 mg/kg, high levels of tumor necrosis factor alpha (TNF-alpha), ceramides, and nuclear factor kappaB accumulated in lung and alveolar macrophages. Both acid and neutral sphingomyelinases were activated within 4 h. These signal transduction events were associated with alteration in the oxygen defense system. Within 1 h of exposure to CEES (6 mg/kg body weight), there was 10-fold increase in the (125)I-BSA leakage into lung tissue, indicating severe lung injury. Although low level of CEES exposure (0.5 mg/kg body weight) produced symptoms of chemical burn in lung as early as 1 h after exposure, the severity of edema, congestion, hemorrhage, and inflammation increased progressively with time (1 h to 21 days). Feeding of single dose of NAC (0.5 g) by gavage just before the CEES infusion was ineffective to counteract these effects. However, consumption of the antioxidant in drinking water for 3 or 30 days prior to CEES exposure significantly inhibited the induction of TNF-alpha, activation of neutral and acid sphingomyelinases, production of ceramides, activation of caspases, leakage of (125)I-bovine serum albumin ((125)I-BSA) into lung tissue, and histological alterations in lung. Pretreatment with NAC for 3 and 30 days protected against 69-76% of the acute lung injury. Therefore, NAC may be an antidote for CEES-induced lung injury.

Effect of nitroreduction on the alkylating reactivity and cytotoxicity of the 2,4-dinitrobenzamide-5-aziridine CB 1954 and the corresponding nitrogen mustard SN 23862: distinct mechanisms of bioreductive activation.

The dinitrobenzamide aziridine CB 1954 (1) and its nitrogen mustard analogue SN 23862 (6) are prodrugs that are activated by enzymatic nitroreduction in tumors. Bioactivation of 1 is considered to be due to reduction of its 4-nitro group to the hydroxylamine and subsequent formation of the N-acetoxy derivative; this acts as a reactive center, in concert with the aziridine moiety, to provide a bifunctional DNA cross-linking agent (Knox model). It is currently unclear whether bioactivation of 6 occurs by the same mechanism or results from the electronic effects of nitroreduction on reactivity of the nitrogen mustard moiety. To discriminate between these mechanisms, we have synthesized the hydroxylamine and amine derivatives of 1 and 6, plus related compounds, and determined their alkylating reactivities in aqueous solution, using LC/MS to identify reaction pathways. The relationships between substituent electronic effects, reactivity, and cytotoxicity were determined using the UV4 cell line, which is defective in nucleotide excision repair (thus avoiding differences in repair kinetics). Alkylating reactivity correlated with the electron-donating character of the ortho or para substituent in the case of the mustards, with a less marked electronic effect for the aziridines. Importantly, there was a highly significant linear relationship between cytotoxic potency and alkylating reactivity in both the aziridine and the mustard series, with the notable exception of 4, the 4-hydroxylamine of 1, which was 300-fold more toxic than predicted by this relationship. This demonstrates that the high potency of 4 does not result from activation of the aziridine ring, supporting the Knox model. The single-step bioactivation of 6, to amino or hydroxylamine metabolites with similar potency to 4, is a potential advantage in the use of dinitrobenzamide mustards as prodrugs for activation by nitroreductases.

Signal transduction events in lung injury induced by 2-chloroethyl ethyl sulfide, a mustard analog.

Sulfur mustard has been used as a vesicant chemical warfare agent. To understand the mechanism by which mustard gas exposure causes respiratory damage, we have used 2-chloroethyl ethyl sulfide (CEES) as a mustard analog. Our initial studies have shown that guinea pigs exposed to CEES intratracheally accumulate high levels of TNF-alpha. Accumulation of TNF-alpha leads to activation of both acid and neutral sphingomyelinases, resulting in high accumulation of ceramides, a second messenger involved in cell apoptosis. In addition, NF-kappa B was activated for a short period (1-2 h after exposure) as determined by mobility shift assay. Supershift assays indicated that both p50 and p65 of NF-kappa B were activated due to CEES exposure. However, NF-kappa B rapidly disappeared after 2 h. It is possible that the initial activation of NF-kappa B was an adaptive response to protect the cells from damage since NF-kappa B is known to inhibit TNF-alpha/ceramide-induced cell apoptosis. Since NF-kappa B disappeared after 2 h, the cells continued being damaged owing to accumulation of ceramides and activation of several caspases, leading to apoptosis.

Isophosphoramide mustard analogues as prodrugs for anticancer gene-directed enzyme-prodrug therapy (GDEPT).

Two types of prodrugs, benzyl analogues of isophosphoramide mustard (iPAM), activated by cytochrome P450, and acylthioethyl analogues, activated by esterases, were designed. In contrast to iPAM that hydrolyse rapidly, the examined compounds are stable in phosphate-buffered saline and Tris buffer. Benzyl analogues of iPAM are poor substrates for cytochrome P450, are not cytotoxic and posses no antitumour activity. Acylthioethyl analogues of iPAM are good substrates for pig liver esterase, are cytotoxic and exert antitumour activity against L1210 leukaemia in mice. The observed correlation for iPAM analogues between their susceptibility to hydrolysis and cytotoxicity and antitumour activity suggests possible application of these compounds as the prodrugs in gene-directed enzyme-prodrug therapy.

Chromatin structure at the 3′-boundary of the human β-globin locus control region hypersensitive site-2

Chromatin structure was examined at the 3′-boundary region of the human β-globin locus control region hypersensitive site-2 (LCR HS-2) using several footprinting agents. Erythroid K562 cells (possessing HS-2) were damaged by the footprinting agents: hedamycin, bleomycin and four nitrogen mustard analogues. Purified DNA and non-erythroid HeLa cells (lacking HS-2) were also damaged as controls for comparison with K562 cells. The comparison between intact cells and purified DNA showed several protected regions in K562 cells. A large erythroid-specific protected region of 135 bp was found at the boundary of HS-2. The length of this protected region (135 bp) was close to that of DNA contained in a nucleosome core (146 bp). Another two protected regions were found upstream of the protected region. A 16-bp erythroid-specific footprint co-localised with a GATA-1 motif—this indicated that the GATA-1 protein could be involved in positioning the nucleosome. Further upstream, a 100-bp footprint coincided with an AT-rich region. Thus our footprinting results suggest that the 3′-boundary of LCR HS-2 is flanked by a positioned nucleosome and that an erythroid-specific protein binds to the sequence adjacent to the nucleosome and acts to position the nucleosome at the boundary of the hypersensitive site.

Recent developments in sequence selective minor groove DNA effectors.

DNA is a well characterized intracellular target but its large size and sequential nature make it an elusive target for selective drug action. Binding of low molecular weight ligands to DNA causes a wide variety of potential biological responses. In this respect the main consideration is given to recent developments in DNA sequence selective binding agents bearing conjugated effectors because of their potential application in diagnosis and treatment of cancers as well as in molecular biology. Recent progress in the development of cross linked lexitropsin oligopeptides and hairpins, which bind selectively to the minor groove of duplex DNA, is discussed. Bis-distamycins and related lexitropsins show inhibitory activity against HIV-1 and HIV-2 integrases at low nanomolar concentrations. Benzoyl nitrogen mustard analogs of lexitropsins are active against a variety of tumor models. Certain of the bis-benzimidazoles show altered DNA sequence preference and bind to DNA at 5'CG and TG sequences rather than at the preferred AT sites of the parent drug. A comparison of bifunctional bizelesin with monoalkylating adozelesin shows that it appears to have an increased sequence selectivity such that monoalkylating compounds react at more than one site but bizelesin reacts only at sites where there are two suitably positioned alkylation sites. Adozelesin, bizelesin and carzelesin are far more potent as cytotoxic agents than cisplatin or doxorubicin. A new class of 1,2,9,9a-tetrahydrocyclo-propa[c]benz[e]indole-4-one (CBI) analogs i.e., CBI-lexitropsin conjugates arising from the latter leads are also discussed.A number of cyclopropylpyrroloindole (CPI) and CBI-lexitropsin conjugates related to CC-1065 alkylate at the N3 position of adenine in the minor groove of DNA in a sequence specific manner, and also show cytotoxicities in the femtomolar range. The cross linking efficiency of PBD dimers is much greater than that of other cross linkers including cisplatin, and melphalan. A new class of PBD-lexitropsin conjugates is also discussed. Certain functional models of the bleomycins (BLMs) show outstanding DNA cleavage activity comparable with that of and positionally distinct from natural BLM.

Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents

There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites, in addition to sites characteristic of untargeted mustards.

Metabolism and Pharmacokinetics of Oxazaphosphorines

The 2 most commonly used oxazaphosphorines are cyclophosphamide and ifosfamide, although other bifunctional mustard analogues continue to be investigated. The pharmacology of these agents is determined by their metabolism, since the parent drug is relatively inactive. For cyclophosphamide, elimination of the parent compound is by activation to the 4-hydroxy metabolite, although other minor pathways of inactivation also play a role. Ifosfamide is inactivated to a greater degree by dechloroethylation reactions. More robust assay methods for the 4-hydroxy metabolites may reveal more about the clinical pharmacology of these drugs, but at present the best pharmacodynamic data indicate an inverse relationship between plasma concentration of parent drug and either toxicity or antitumour effect. The metabolism of cyclophosphamide is of particular relevance in the application of high dose chemotherapy. The activation pathway of metabolism is saturable, such that at higher doses (greater than 2 to 4 g/m2) a greater proportion of the drug is eliminated as inactive metabolites. However, both cyclophosphamide and ifosfamide also act to induce their own metabolism. Since most high dose regimens require a continuous infusion or divided doses over several days, saturation of metabolism may be compensated for, in part, by auto-induction. Although a quantitative distinction may be made between the cytochrome P450 isoforms responsible for the activating 4-hydroxylation reaction and those which mediate the dechloroethylation reactions, selective induction of the activation pathway, or inhibition of the inactivating pathway, has not been demonstrated clinically. Mathematical models to describe and predict the relative contributions of saturation and autoinduction to the net activation of cyclophosphamide have been developed. However, these require careful validation and may not be applicable outside the exact regimen in which they were derived. A further complication is the chiral nature of these 2 drugs, with some suggestion that one enantiomer may have a favourable profile of metabolism over the other. That the oxazaphosphorines continue to be the subject of intensive investigation over 30 years after their introduction into clinical practice is partly because of their antitumour activity. Further advances in analytical and molecular pharmacological techniques may further optimise their use and allow rational design of more selective analogues.

Protein–DNA footprinting of the human ϵ-globin promoter in human intact cells using nitrogen mustard analogues and other DNA-damaging agents

Nitrogen mustard analogues, bleomycin and dimethyl sulphate (DMS) have been used as probes of protein–DNA interactions in intact human cells. The sites of damage have been determined at base pair resolution in the single copy ϵ-globin gene promoter in erythroid K562 cells, non-erythroid HeLa cells and purified DNA. Exponential amplification of gene-specific damage fragments was achieved using the ligation-mediated polymerase chain reaction (LMPCR) technique and analysed on DNA sequencing gels. A comparison of the relative damage band intensities between purified DNA and intact cells revealed several significant differences – both protection (footprint) and enhancement. These differences occurred at putative transcription factor binding sites and hence are thought to be due to protein–DNA interactions. A major feature of the band intensity ratio plots was the footprint observed at the CCAAT box binding motif as revealed by nitrogen mustard analogues. Enhanced band intensity (hypersensitivity) was displayed at the 5′- and 3′-ends of the CCAAT box in K562 cells – this feature was absent in HeLa cells and in vitro reconstitutions. A footprint was found at the GATA-1 motif in K562 cells that was also absent in non-expressing HeLa cells. Footprints were also evident at the TATA box, CACC box and the ϵF1 DNA binding motif in K562 cells.

Synthesis of antineoplaston A10 analogs as potential antitumor agents.

Several aniline mustard analogues were obtained by introducing N,N-bis(2-chloroethyl)amino moiety to phenyl ring of A10 analogues in order to increase reactivity of A10 analogs and selectivity into DNA. The in vitro antitumor activity of synthesized compounds was evaluated using five different solid tumor cell lines by SRB method. Aniline mustard analogues exhibited more potent antitumor activity than A10 analogs. Especially, m-aniline mustard of benzoyl analogue displayed remarkable antitumor activity.