2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT.

An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours. Activation of the aziridinyl dinitrobenzamide CB 1954 by E. coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite. We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2- and 4-hydroxylamines and their corresponding amines). The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines. Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line. These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours. The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites. These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.

Sensitive liquid chromatographic assay for the basic DNA intercalator (N,N-dimethylaminoethyl)-9-amino-5-methylacridine-4-carboxamide and its nitroarylmethyl quaternary prodrugs in biological samples.

Nitroarylmethyl quaternary (NMQ) ammonium salts of the basic DNA intercalator AMAC (N,N-dimethylaminoethyl-9-amino-5-methylacridine-4-carboxamide) are of interest as anticancer prodrugs. A sensitive HPLC assay has been developed for quantitation of AMAC and its NMQ prodrugs in cultured cells, plasma and tissue. Recovery of the prodrugs, without conversion to AMAC, was achieved using extraction in alkaline acetonitrile followed by immediate reneutralisation. Reversed-phase HPLC with fluorescence detection gave a detection limit of 3 fmol for AMAC, with linearity to 20 nmol (using diode array absorbance at high concentrations). This assay was used to measure cellular uptake, and hypoxic metabolism to AMAC, of three NMQ-AMAC prodrugs.

Reduction of nitroarylmethyl quaternary ammonium prodrugs of mechlorethamine by radiation.

Nitroarylmethyl quaternary ammonium nitrogen mustards are bioreductive drugs designed to release mechlorethamine, when reduced metabolically, via fragmentation of the initial nitro radical anion to a benzylic-type radical. This proposed mechanism (termed Type I) is analogous to the well-known reductive fragmentation of 2-nitrobenzyl halides. The lead nitroarylmethyl quaternary mustard SN 25246 (NSC 656581), which contains a 2-nitrobenzyl electron acceptor, was shown previously to release mechlorethamine in hypoxic cell cultures and to be a highly selective hypoxic cytotoxin. In the present work the mechanism of reductive release of mechlorethamine from nitroarylmethyl quaternary prodrugs was investigated by steady-state radiolysis with product analysis by high-performance liquid chromatography. SN 25246 releases mechlorethamine in high yield upon reduction, but several reducing equivalents are required (Type II mechanisms). Investigation of other nitroarylmethyl units identified two heterocyclic analogues, the 1-methyl-4-nitro-5-imidazolyl derivative SN 25341 and the 1-methyl-5-nitro-2-pyrrolyl derivative SN 26581, which have a reduction stoichiometry of about one reducing equivalent and which release mechlorethamine efficiently. The other products from reduction of SN 25341 are also consistent with Type I fragmentation, via intramolecular electron transfer, to give the 1-methyl-4-nitroimidazole-5-CH2. radical. The sensitivity of the 4-nitroimidazole and 5-nitropyrrole nitroarylmethyl quaternary mustards to Type I reductive fragmentation suggests that these electron acceptor units may be well suited to development of prodrugs which release tertiary amine effectors after metabolic or radiolytic reduction in hypoxic regions of tumors.

Hypoxia-selective antitumor agents. 13. Effects of acridine substitution on the hypoxia-selective cytotoxicity and metabolic reduction of the bis-bioreductive agent nitracrine N-oxide.

A series of nuclear-substituted derivatives of nitracrine N-oxide (2; a bis-bioreductive hypoxia-selective cytotoxin) were prepared and evaluated, seeking analogues of lower nitroacridine reduction potential. Disubstitution with Me or OMe groups at the 4- and 5-positions did not provide analogues with one-electron reduction potentials significantly lower than those of the corresponding monosubstituted derivatives (E(1) ca. -350 mV for both the 4-OMe and 4,5-diOMe compounds). This appears not to be due to a concomitant raising of the acridine pKa but to a lack of direct electronic effect of substituents in the ring not bearing the nitro group. Conversely, placing two OMe groups in the nitro-bearing ring does result in a substantial further lowering of reduction potential (the 2,4-diOMe analogue has an E(1) of -401 mV). The mono- and disubstituted N-oxides have substantially lower cytotoxicities than the parent nitracrine N-oxide 2 but generally retain very high hypoxic selectivity. The OMe-substituted N-oxides all showed greater metabolic stability than 2 in hypoxic AA8 cell cultures, and the 4-OMe compound 6 had improved activity in EMT6 multicellular spheroids suggesting that this metabolic stabilization may allow more efficient diffusion in tumor tissue. The parent compound 2 was selectively toxic to hypoxic cells in KHT tumors in vivo and clearly superior to nitracrine itself (although only at doses which would eventually be lethal to the host). The analogues of lower E(1), including 6, were not superior to 2 in vivo, indicating that metabolic stabilization of the nitro group is not alone sufficient to improve therapeutic utility.

Anti-inflammatory effects of LPS, MDP and FMLP on carrageenan pleurisy in the rat.

Bacterial products fmet-leu-phe (FMLP), muramyl dipeptide (MDP) and lipopolysaccharide (LPS) were assayed for their ability to alter the inflammatory response to lambda carrageenan-induced pleurisy in Hooded Surgery rats. Continuously infused FMLP, or one initial i.v. dose of FMLP, MDP or LPS either ablated or partially suppressed the pleurisy. Total circulating leucocytes and neutrophils were suppressed by 55-65% when compared to the normal circulating leucocyte response to carrageenan pleurisy, excepting the protocol incorporating a single i.v. dose of FMLP where suppression was intermediate at 30%. There were also significant changes in the expression of FMLP receptors on circulating neutrophils. MDP and LPS induced a receptor number increase of 2 and 1.7 times initial value respectively, whilst a continuous FMLP infusion caused a receptor decrease to 0.3 times the initial value. The introduction of bacterial products at an alternative site to that of the pleurisy had an anti-inflammatory effect and the pleurisy was reduced.

Formylmethionyl-leucylphenylalanine and the SOS operon in Escherichia coli: a model of host-bacterial interactions.

To determine the biological significance of the existence of highly specific receptors for the bacterial chemotactic peptide formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) on neutrophil leucocytes, we investigated the role of this peptide in bacterial metabolism. The UmuD protein of the Escherichia coli SOS operon was identified as having an N-terminal fMet-Leu-Phe sequence and a recombinant E. coli with the umuD gene on plasmid pSB13 was shown to be an over-producer of both UmuD and fMet-Leu-Phe. Activation of SOS genes in conventional wild-type E. coli (K12) by u.v. light or hydrogen peroxide increased fMet-Leu-Phe production up to 4-fold. A RecA- strain, incapable of SOS activation, was a low basal producer of fMet-Leu-Phe and showed no increased production with u.v. light or oxidant stress. We propose that host phagocytes respond to fMet-Leu-Phe and closely related peptides because they are generated by bacteria under oxidant stress. Increased fMet-Leu-Phe production may signal to the host a change in the organism's biological status from commensal to pathogen because of the invasion into tissues exposing bacteria to high pO2 levels and oxidant stress.

Assessment of neutrophil leukocyte secretory response to fMLP in whole blood in vitro.

A simple, precise method has been developed for assessing neutrophil secretory responses (release of vitamin B12 binding protein from specific granules) to challenge of aliquots of whole blood with the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Dose-response studies performed on blood from normal healthy volunteers showed higher maximal secretory responses in males than females (33.3 +/- SEM 2.2 vs. 27.4 +/- 2.5, P less than .005) a left shift in dose-response curves after feeding compared to fasting (P less than .005), spontaneous up-regulation of responses in blood incubated at 37 degrees C for 1 h, and marked upregulation in response to preincubation with endotoxin. This whole blood challenge method may be used to study neutrophil responses in groups of individuals or patients without the confounding effects of changes in cell responses resulting from cell isolation procedures. The method may also be used as a bioassay for neutrophil-activating factors.

Measurements of unsaturated vitamin B12-binding capacity and myeloperoxidase as indices of severity of acute inflammation in serial colonoscopy biopsy specimens from patients with inflammatory bowel disease.

Measurements of tissue content of myeloperoxidase, a constituent of neutrophil azurophil granules and of unsaturated vitamin B12-binding protein from neutrophil-specific granules, have been used to assess intestinal inflammation. This paper reports results of a prospective evaluation of such measurements in serial colonoscopy biopsy specimens from patients with inflammatory bowel disease. Histologic grading of acute inflammation was based on perceived numbers of neutrophil polymorphs in sections from an immediately adjacent biopsy specimen. The mean + 2 SD range for unsaturated vitamin B12-binding protein activity in homogenates of histologically normal specimens was 62 pg mg protein-1. Values increased progressively up to 900 pg mg-1 protein in the most severely inflamed specimens. Unsaturated vitamin B12-binding protein measurements generally distinguished among histologic grades of inflammation, whereas myeloperoxidase activities failed to do this, probably because substantial myeloperoxidase activity was found in uninflamed colonic mucosa, suggesting a non-neutrophil source for this enzyme.

Bacterial chemotactic oligopeptides and the intestinal mucosal barrier.

Intestinal absorption and enterohepatic circulation of N-formyl-methionyl-leucyl-125I-tyrosine, a bioactive synthetic analog of the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine has been investigated in the rat. In ileum and proximal and distal colon, dithiothreitol, which increases mucosal permeability, increased peptide absorption and biliary recovery fourfold, 70-fold, and 20-fold over control values, respectively. When dithiothreitol was combined with d-l-benzyl succinate, a potent inhibitor of intestinal carboxypeptidase, absorption and biliary recovery from ileal loops increased markedly to 40-fold over control, whereas there was no further increase in absorption from colon loops. There was a strong correlation between biliary N-formyl-methionyl-leucyl-125I-tyrosine recovery and intestinal absorption of 51Cr-ethylenediaminetetraacetate, a marker of passive mucosal permeability (r = 0.97). We conclude that in the ileum both enzymic degradation and restricted mucosal permeability contribute to the intestinal barrier to luminal bacterial formyl oligopeptides. In the colon, however, enzymic mechanisms are less active and restricted mucosal permeability is the major factor. Abnormalities of the intestinal mucosal barrier to proinflammatory bacterial peptides could play a role in inflammatory disorders of the gut.

Enterohepatic circulation of bacterial chemotactic peptide in rats with experimental colitis.

The association of hepatobiliary disorders with colonic inflammation is well recognized. Although the pathophysiology is obscure, increased permeation of toxic bacterial products across the inflamed gut to the portal circulation might be one mechanism. Potentially toxic metabolites include N-formylated chemotactic peptides that are produced by several species of intestinal bacteria and can be detected in colonic fluid in vivo. To investigate the metabolic fate of one of these low molecular weight proinflammatory peptides, N-formyl L-methionine L-leucine 125I-L-tyrosine was introduced into colon loops of healthy rats (n = 10) and rats with experimental colitis (n = 15) induced by rectal instillation of 15% (vol/vol) acetic acid. Gut, liver, and blood radioactivity were monitored by external gamma-counting and radioactivity in bile was measured by biliary catheter drainage into a well counter. Bile was processed by high-performance liquid chromatography to determine the amount of intact, bioactive peptide excreted over 3 h. After colonic instillation of 1 nmol of peptide, the mean (+/- SEM) biliary excretion of intact peptide was 6.4 +/- 2.0 pmol in normal rats and 49.0 +/- 20 pmol in rats with colitis (p less than 0.01). An enterohepatic circulation of synthetic N-formyl L-methionine L-leucine L-tyrosine has been demonstrated in the rat. Experimental colitis was associated with an eightfold increase in biliary excretion of this proinflammatory bacterial peptide. Proinflammatory bacterial peptides synthesized by colonic bacteria could be important in the pathophysiology of colon inflammation and its frequently associated hepatobiliary complications.