HIF-1α inhibitor PX-478 preserves pancreatic ß cell function in diabetes.

During progression of type 2 diabetes, pancreatic ß cells are subjected to sustained metabolic overload. We postulated that this state mediates a hypoxic phenotype driven by hypoxia-inducible factor-1α (HIF-1α) and that treatment with the HIF-1α inhibitor PX-478 would improve ß cell function. Our studies showed that the HIF-1α protein was present in pancreatic ß cells of diabetic mouse models. In mouse islets with high glucose metabolism, the emergence of intracellular Ca2+ oscillations at low glucose concentration and the abnormally high basal release of insulin were suppressed by treatment with the HIF-1α inhibitor PX-478, indicating improvement of ß cell function. Treatment of db/db mice with PX-478 prevented the rise of glycemia and diabetes progression by maintenance of elevated plasma insulin concentration. In streptozotocin-induced diabetic mice, PX-478 improved the recovery of glucose homeostasis. Islets isolated from these mice showed hallmarks of improved ß cell function including elevation of insulin content, increased expression of genes involved in ß cell function and maturity, inhibition of dedifferentiation markers, and formation of mature insulin granules. In response to PX-478 treatment, human islet organoids chronically exposed to high glucose presented improved stimulation index of glucose-induced insulin secretion. These results suggest that the HIF-1α inhibitor PX-478 has the potential to act as an antidiabetic therapeutic agent that preserves ß cell function under metabolic overload.

Evaluation of amino acid-mustard transport as L-type amino acid transporter 1 (LAT1)-mediated alkylating agents.

The L-type amino acid transporter 1 (LAT1, SLC7A5) is an Na+-independent neutral amino acid transporter the expression of which is located in retinal endothelial cells. Due to its broad substrate selectivity, LAT1 has been proposed to mediate the transport of amino acid-related drugs across the blood-tissue barriers. Here, we have investigated the transport screening of amino acid-mustards using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) which expresses LAT1. We synthesized 5 amino acid-mustards: tyrosine-mustard, phenylglycine-mustard, alanine-mustard, ornithine-mustard, and lysine-mustard. LAT1-mediated [3H]L-phenylalanine (Phe) uptake by TR-iBRB2 cells was inhibited in a competitive manner by tyrosine-mustard and phenylglycine-mustard as well as melphalan (phenylalanine-mustard). Phenylglycine-mustard was able to induce the efflux of [3H]Phe preloaded into the TR-iBRB2 cells expressing LAT1 through the obligatory exchange mechanism, although tyrosine-mustard, alanine-mustard, ornithine-mustard, lysine-mustard, and melphalan did not induce any significant efflux. These findings suggest that phenylglycine-mustard is a better substrate for LAT1 than melphalan and other amino acid-mustards.

Development of a liquid chromatography-multiple reaction monitoring procedure for concurrent verification of exposure to different forms of mustard agents.

A novel liquid chromatography-multiple reaction monitoring (LC-MRM) procedure has been developed for retrospective diagnosis of exposure to different forms of mustard agents. This concise method is able to validate prior exposure to nitrogen mustards (HN-1, HN-2, and HN-3) or sulfur mustard (HD) in a single run, which significantly reduces analysis time compared to separate runs to screen for different mustards' biomarkers based on tandem mass spectrometry. Belonging to one of the more toxic classes of chemical warfare agents, these potent vesicants bind covalently to the cysteine-34 residue of human serum albumin. This results in the formation of stable adducts whose identities were confirmed by a de novo sequencing bioinformatics software package. Our developed technique tracks these albumin-derived adduct biomarkers in blood samples which persist in vitro following exposure, enabling a detection limit of 200 nM of HN-1, 100 nM of HN-2, 200 nM of HN-3, or 50 nM of HD in human blood. The CWA-adducts formed in blood samples can be conveniently and sensitively analyzed by this MRM technique to allow rapid and reliable screening.

Influence of mustard group structure on pathways of in vitro metabolism of anticancer N-(2-hydroxyethyl)-3,5-dinitrobenzamide 2-mustard prodrugs.

The dinitrobenzamide mustards are a class of bioreductive nitro-aromatic anticancer prodrugs, of which a phosphorylated analog (PR-104) is currently in clinical development. They are bioactivated by tumor reductases to form DNA cross-linking cytotoxins. However, their biotransformation in normal tissues has not been examined. Here we report the aerobic in vitro metabolism of three N-(2 hydroxyethyl)-3,5-dinitrobenzamide 2-mustards and the corresponding nonmustard analog in human, mouse, rat, and dog hepatic S9 preparations. These compounds have a range of mustard structures (-N(CH(2)CH(2)X)(2) where X = H, Cl, Br, or OSO(2)Me). Four metabolic routes were identified: reduction of either nitro group, N-dealkylation of the mustard, plus O-acetylation, and O-glucuronidation of the hydroxyethyl side chain. Reduction of the nitro group ortho to the mustard resulted in intramolecular alkylation and is considered to be an inactivation pathway, whereas reduction of the nitro group para to the mustard generated potential DNA cross-linking cytotoxins. N-Dealkylation inactivated the mustard moiety but may result in the formation of toxic acetaldehyde derivatives. Increasing the size of the nitrogen mustard leaving group abrogated the ortho-nitroreduction and N-dealkylation routes and thereby improved overall metabolic stability but had little effect on aerobic para-nitroreduction. All four compounds underwent O-glucuronidation of the hydroxyethyl side chain and further studies to elucidate the relative importance of this pathway in vivo are in progress.

Reactions of {4-[bis(2-chloroethyl)amino]phenyl}acetic acid (phenylacetic acid mustard) with 2'-deoxyribonucleosides.

Phenylacetic acid mustard (PAM; 2), a major metabolite of the anticancer agent chlorambucil (CLB; 1), was allowed to react with 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), 2'-deoxy-5-methylcytidine (dMeC), and thymidine (T) at physiological pH (cacodylic acid, 50% base). The reactions were followed by HPLC and analyzed by HPLC/MS and/or (1)H-NMR techniques. Although the predominant reaction observed was hydrolysis of PAM, 2 also reacted with various heteroatoms of the nucleosides to give a series of products: compounds 5-31. PAM (2) was found to be hydrolytically slightly more stable than CLB (1). The principal reaction sites of 2 with dA, dG, and with all pyrimidine nucleosides were N(1), N(7), and N(3), resp. Also, several other adducts were detected and characterized. There was no significant difference in the reactivity of 1 and 2 with dG, dA or T, but the N(3) dC-PAM adduct was deaminated easier than the corresponding CLB derivative. The role of PAM-2'-deoxyribonucleoside adducts on the cytotoxic and mutagenic properties of CLB (1) is discussed.

Metabolite changes in HT-29 xenograft tumors following HIF-1alpha inhibition with PX-478 as studied by MR spectroscopy in vivo and ex vivo.

The hypoxia-inducible transcription factor (HIF-1alpha) plays a central role in tumor development. PX-478 is an experimental anti-cancer drug known to inhibit HIF-1alpha in experimental tumors. The purpose of this study was to identify MRS-visible metabolic biomarkers for PX-478 response prior to phase I/II clinical trials. Single-voxel in vivo localized (1)H spectra were obtained from HT-29 tumor xenografts prior and up to 24 h after treatment with a single dose of PX-478. Profiles of water-soluble and lipophilic metabolites were also examined ex vivo with both (1)H and (31)P spectroscopy for peak identification and to interrogate the underlying biochemistry of the response. The total choline (tCho) resonance was significantly decreased in vivo 12 and 24 h following treatment with PX-478 and this was confirmed with high-resolution (1)H and (31)P MRS. In non-aqueous extracts, significant reductions in cardiolipin, PtdEtn (phosphatidylethanolamine) and PtdI (phosphatidylinositol) were seen in response to PX-478. Although there were trends to a decrease in lactate (and lipid) resonances in vivo and ex vivo, these changes were not significant. This is in contrast to inhibition of in vitro glucose consumption and lactate production by PX-478 in HT-29 cells. The significant and robust change in tCho has identified this as a potential (1)H MRS-visible biomarker for drug response in vivo while high-resolution spectroscopy indicated that GPC, PC, myoI, PE, GPE, CL, PtdEtn and PtdI are potential ex vivo response biomarkers.

Novel fluorinated prodrugs for activation by carboxypeptidase G2 showing good in vivo antitumor activity in gene-directed enzyme prodrug therapy.

Sixteen novel polyfluorinated benzoic acid mustards have been synthesized for use in gene-directed enzyme prodrug therapy (GDEPT). Eight of these were benzoic acid L-glutamate mustards for evaluation as prodrugs and the other eight were the active drugs formed by the action of the bacterial enzyme carboxypeptidase G2 (CPG2). All of the di- and trifluorinated prodrugs were efficiently cleaved by the enzyme. In contrast, the tetrafluorinated prodrugs were found to be competitive inhibitors of CPG2, the first such inhibitors to have been described. The di- and trifluorinated prodrugs were differentially cytotoxic to human breast carcinoma cells (MDA MB 361) expressing CPG2, compared to control cells that did not express the enzyme. The difluorinated prodrug {4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoyl}-L-glutamic acid and its iodoethylamino analogue were effective substrates for the enzyme and showed excellent therapeutic activity in CPG2-expressing MDA MB 361 xenografts, either curing or greatly inhibiting tumor growth and extending the life of the animals.

Distamycin A as stem of DNA minor groove alkylating agents.

Analogues of naturally occurring antitumor agents, such as distamycin A, which bind in the minor groove of DNA, represent a new class of anticancer compounds currently under investigation. Distamycin A has driven researcher's attention not only for their biological activity, but also for its non intercalative binding to the minor groove of double-stranded B-DNA, where it forms strong reversible complex preferentially at the nucleotide sequences consisting of 4-5 adjacent AT base pairs. The pyrrole-amide skeleton of distamycin A has been also used as DNA sequence selective vehicles for the delivery of alkylating functions to DNA targets, leading to a sharp increase of its cytotoxicity, in comparison to that, very weak, of distamycin itself. In the last few years, several hybrid compounds, in which known antitumor derivatives or simple active moieties of known antitumor agents have been tethered to distamycin frames, have been designed, synthesized and tested. Several efforts have been made to modify DNA sequence selectivity and stability of the distamycin and the structural modifications have been based on replacement of pyrrole by other heterocycles and/or benzoheterocycles obtaining a novel class of minor groove binding molecules called lexitropsins. The role of the amidino moiety, by means of the substitution with various groups, which includes ionizable, acid or basic, and non-ionizable groups, has been also studied. The synthesis of a hybrid deriving among the combination of the distamycin A and naturally occurring alkylating agent has been also reported. Several classes of distamycin derivatives that have been reported in the published literature have been described in this review article.

Interaction of oligonucleotides with cellular proteins.

Oligonucleotides (ODNs) conjugated to 4-[(N-2-chloroethyl-N-methyl)amino] benzylamine were used to investigate ODN-binding proteins in cells of different origin. The data obtained demonstrate that 68, 46, 38 and 28 kDa ODN-binding proteins are universal for tested cell lines.

Rapid determination of the anti-cancer drug chlorambucil (Leukeran) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A bioanalytical method for the determination of the anticancer drug chlorambucil (Leukeran) and its phenyl acetic acid mustard metabolite in human serum and plasma is described. Automated solid-phase extraction of the analytes is carried out with C18 sorbent packed in a 96 well format microtitre plate using a robotic sample processor. The extracts are analysed by isocratic reversed-phase liquid chromatography using pneumatically and thermally assisted electrospray ionisation (TurboIonspray) with selected reaction monitoring. The method is specific and sensitive, with a range of 4-800 ng/ml in human serum and plasma for both parent drug and metabolite (sample volume 200 microl). The method is accurate and precise with intra-assay and inter-assay precision (C.V.) of <15% and bias <15% for both analytes. The automated extraction procedure is significantly faster than manual sample pre-treatment methods, a batch of 96 samples is extracted in 50 min allowing for faster sample turnaround. The method has been used to provide pharmacokinetic support to biocomparability studies of Leukeran following single doses of oral tablet formulations.

DNA minor groove targeted alkylating agents based on bisbenzimidazole carriers: synthesis, cytotoxicity and sequence-specificity of DNA alkylation.

A series of bisbenzimidazoles bearing a variety of alkylating agents [ortho- and meta-mustards, imidazolebis(hydroxymethyl), imidazolebis(methylcarbamate) and pyrrolebis(hydroxymethyl)], appended by a propyl linker chain, were prepared and investigated for sequence-specificity of DNA alkylation and their cytotoxicity. Previous work has shown that, for para-aniline mustards, a propyl linker is optimal for cytotoxicity. Alkaline cleavage assays using a variety of different labelled oligonucleotides showed that the preferred sequences for adenine alkylation were 5'-TTTANANAANN and 5'-ATTANANAANN (underlined bases show the drug alkylation sites), with AT-rich sequences required on both the 5' and 3' sides of the alkylated adenine. The different aniline mustards showed little variation in alkylation pattern and similar efficiencies of DNA cross-link formation despite the changes in orientation and positioning of the mustard, suggesting that the propyl linker has some flexibility. The imidazole- and pyrrolebis(hydroxymethyl) alkylators showed no DNA strand cleavage following base treatment, indicating that no guanine or adenine N3 or N7 adducts were formed. Using the PCR-based polymerase stop assay, these alkylators showed PCR blocks at 5'-C*G sites (the * nucleotide indicates the blocked site), particularly at 5'-TAC*GA 5'-AGC*GGA, and 5'-AGCC*GGT sequences, caused by guanine 2-NH2 lesions on the opposite strand. Only the (more reactive) imidazolebis(methylcarbamoyl) and pyrrolebis(hydroxymethyl) alkylators demonstrated interstrand cross-linking ability. All of the bifunctional mustards showed large (approximately 100-fold) increases in cytotoxicity over chlorambucil, with the corresponding monofunctional mustards being 20- to 60-fold less cytotoxic. These results suggest that in the mustards the propyl linker provides sufficient flexibility to achieve delivery of the alkylator to favoured (adenine N3) sites in the minor groove, regardless of its exact geometry with respect to the bisbenzimidazole carrier. The 'targeted' bisbenzimidazole bis(hydroxymethyl)pyrrole- and imidazole analogues showed very similar patterns of alkylation to the corresponding 'untargeted' compounds, with little evidence of additional selectivity imposed by this AT-preferring carrier.

Synthesis of self-immolative glucuronide-based prodrugs of a phenol mustard.

The design and synthesis of the mustard pro-prodrugs which can be used in ADEPT is reported. Prodrugs 1 and 2 include a glucuronide group which is connected to the drug via an aromatic and/or aliphatic bis-carbamate spacer. The design of these new prodrugs takes advantage of a spontaneous 1,6-elimination and/or an intramolecular cyclization reaction after enzymatic cleavage. Thus, enzymatic-catalyzed hydrolysis of the glucuronyl moiety of 1 by Escherichia coli beta-glucuronidase results in the liberation of the parent mustard drug 20 with formation of CO2, 2-nitro-4-hydroxymethylphenol 19 and dimethylimidazolidinone 21. Surprisingly, prodrug 2 was not cleaved under the same conditions. According to in vitro experiments, prodrugs 1 and 2 were approximately 50- and 80-fold less cytotoxic than the parent drug and, when treated with beta-glucuronidase, the level of cytotoxic activity of 1 became comparable to that of the drug. Stability of 1 in phosphate buffer was satisfactory. These results demonstrate that 1 is a prodrug that can be specifically activated to release the cytotoxic agent.

Prodrug strategies based on intramolecular cyclization reactions.

Several new prodrug systems for amines, alcohols, and peptides are reviewed. The design of these new prodrug systems takes advantage of several facile intramolecular cyclization reactions, that permit separate manipulation of the release kinetics independent of the structural features of the drug moiety. Such systems can be used for the preparation of esterase-, phosphatase-, and redox-sensitive prodrugs of amines and alcohols and esterase-sensitive cyclic prodrugs of peptides and peptide mimetics.

Design, synthesis, and evaluation of latent alkylating agents activated by glutathione S-transferase.

In search of compounds with improved specificity for targeting the important cancer-associated P1-1 glutathione S-transferase (GST) isozyme, new analogs 4 and 5 of the previously reported glutathione S-transferase (GST)-activated latent alkylating agent gamma-glutamyl-alpha-amino-beta-[[[2-[[bis[bis(2-chloroethyl)amino]ph osp horyl]oxy]ethyl]sulfonyl]propionyl]-(R)-(-)-phenylglycine (3) have been designed, synthesized, and evaluated. One of the diastereomers of 4 exhibited good selectivity for GST P1-1. The tetrabromo analog 5 of the tetrachloro compound 3 maintained its specificity and was found to be more readily activated by GSTs than 3. The GST activation concept was further broadened through design, synthesis, and evaluation of a novel latent urethane mustard 8 and its diethyl ester 9. Interestingly, 8 showed very good specificity for P1-1 GST. Cell culture studies were carried out on 4, 5, 8, and 9 using cell lines engineered to have varying levels of GST P1-1 isozyme. New analogs 4 and 5 exhibited increased toxicity to cell lines with overexpressed GST P1-1 isozyme. The urethane mustard 8 and its diethyl ester 9 were found to be not as toxic. However, they too exhibited more toxicity to a cell line engineered to have elevated P1-1 levels, which was in agreement with the observed in vitro specificity of 8 for P1-1 GST isozyme. Mechanistic studies on alkaline as well as enzyme-catalyzed decomposition of latent mustard 3 provided experimental proof for the hypothesis that 3 breaks down into an active phosphoramidate mustard and a reactive vinyl sulfone. The alkylating nature of the decomposition products was further demonstrated by trapping those transient species as relatively stable diethyldithiocarbamic acid adducts. These results substantially extend previous efforts to develop drugs targeting GST and provide a paradigm for development of other latent drugs.

Design, synthesis and biological evaluation of benzoic acid mustard derivatives of imidazole-containing and C-terminal carboxamide analogues of distamycin.

The synthesis, DNA binding and biological evaluation of two benzoic acid mustard derivatives of imidazole-containing analogues of distamycin in which the C-terminus is modified to contain a terminal carboxamide are described. The apparent DNA binding constants of compounds 5 and 6 were determined using an ethidium displacement assay, and the results showed that they do not have the AT sequence selectivity of distamycin and they show an acceptance for GC base pairs. Based on their pronounced binding to T4 DNA the data suggest that they bind to the minor groove of DNA. The cytotoxicities of compounds 5 and 6 in human chronic myeloid leukemia cells were determined using a MTT assay, and their IC50 values were 27 and 16 microM, respectively, and higher than the corresponding non-terminal carboxamide-containing analogues 3 and 4. Both compounds were however markedly more active than the non-targeted mustard BAM [N,N-bis (-2-chloroethyl)-4-aminobenzoic acid]. In the NCI panel of cell lines 5 gave a distinctly different pattern of tumor selectivity from 6. While these compounds were shown to alkylate DNA using a CD alkylation assay (35 +/- 10% for 5 and 85 +/- 10% for 6), they produced interstrand crosslinks poorly, even at 100 microM drug concentrations. Based on preliminary data from a polymerase stop assay compounds 3-6 gave different patterns of sequence selection monoalkylation which may contribute to their differing biological activities.

[Oligonucleotide-binding proteins of Drosophila: tissue specificity]. / Oligonukleotidsviazyvaiushchie belki Drozofily: tkanevaia spetsifichnost'.

A reaction of native Drosophila proteins with an alkylating oligonucleotide derivative bearing 4-[(N-2-chlorethyl-N-methyl)amino]benzylamine at the 5' terminal phosphate has been investigated. It was found, that the reagent alkylates a few proteins (90, 50, 44, 39, 32 kDa). The modification was organ specific. The labeled 39 kDa protein is present in the ovaries only, while the modified 32 kDa protein is found only in the bulbus.

The use of chemical weapons. Conducting an investigation using survey epidemiology.

The use of chemical weapons in conflict represents a breach of international law as well as a grave violation of human rights. Investigating allegations of their use often is difficult. A basic tool is the survey interview. Experience has shown that a rigorous epidemiologic approach should be taken. A primary emphasis should be designing the study so that consistency of responses can be analyzed to judge the validity of the testimony. Only when the testimony can withstand this scrutiny is it possible to surmise the possible identity of agents employed. Securing samples of the putative agent is of obvious importance. Two recent investigations are discussed herein, one conducted by US Army medical researchers on allegations of chemical weapons use against the Hmong in Laos and another mounted by us on allegations of poison-gas attack against the Iraqi Kurds.

[Chemical transformation of radioactive 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-[32P]phosphamides of oligodeoxyribonucleotides during in vivo experiments]. / Khimicheskie prevrashcheniia radioaktivnykh 4-(N-2-khlorétil-N-metilamino)benzil-5'-[32P]fosfamidov oligodezoksiribonukleotidov pri postanovke éksperimentov in vivo.

The practical use of reactive oligonucleotide derivatives for complementarily addressed modification of nucleic acids in vivo includes several steps, at which side chemical reactions resulting in a decrease of the modification efficiency may take place. Chemical reactions of 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-[32P]phosphamides of oligodeoxyribonucleotides were studied in vivo. The intermolecular self-alkylation at the reactive residue of the alkylating derivative was found in the precipitate of its lithium salt under acetone at-20 degrees C. The effects of pH, buffer solutions, salts, temperature, phenol, cell culture suspensions, tissue homogenates, etc., on the stability of the derivatives were studied. A sufficient cleavage of the phosphamide bond was observed at pH less than 3. In fresh liver homogenates the nucleolytic degradation of the oligonucleotide part of the reagent was shown to occur. After intraperitoneal injection of mice with radioactive alkylating derivatives up to 50% of the reagent was included into the blood biopolymers within one hour. The covalently linked to the biopolymers oligonucleotide appeared to be highly degraded thereby.

Analysis of thiodiglycol in urine of victims of an alleged attack with mustard gas.

A procedure for the semi-quantitative determination of thiodiglycol, a metabolite of the vesicant mustard gas, in urine has been developed. Thiodiglycol was converted into mustard gas using concentrated HCl at temperatures close to 100 degrees C. The headspace of the solution containing mustard gas, was trapped on an adsorption tube filled with Tenax-GC which was subsequently analyzed by gas chromatography/mass spectrometry. Using 10 mL of urine, a detection limit of a few ng/mL of thiodiglycol was achieved. The procedure was applied to urine samples obtained from Iranian patients who were the alleged victims of an attack by chemical warfare agents (probably mustard gas). A number of control samples were investigated as well. Thiodiglycol was found in the urine of the Iranian patients in concentrations varying between 3 and 140 ng/mL. However, the detection of thiodiglycol in concentrations up to 55 ng/mL in control samples excluded the unambiguous verification of the use of mustard gas against the Iranian patients.