The hollow fibre model in cancer drug screening: the NCI experience.

The in vivo hollow fibre model was developed by the National Cancer Institute (NCI) in the United States of America (USA) at a time when the number of potential anti-cancer drugs arising from in vitro screening efforts exceeded the available capacity for testing in traditional xenograft models. Updated analysis of the predictive value of the hollow fibre model continues to indicate that the greater the response in the hollow fibre assay, the more likely it is that activity will be seen in subsequent xenograft models. The original 12 cell line hollow fibre panel has been supplemented with histology-specific panels, and we begin here to analyse their utility in predicting activity in subsequent in vivo models. The key goal of using the hollow fibre model as a way to decrease the cost, both financial and in the number of animals used, to evaluate initial evidence of a compound's capacity to act across physiological barriers continues to be reinforced with our enlarging experience.

A potential role for imaging technology in anticancer efficacy evaluations.

The introduction of imaging methods suitable for rodents offers opportunities for new anticancer efficacy models. Traditional models do not provide the level of sensitivity afforded by these precise and quantitative techniques. Bioluminescent endpoints, now feasible because of sensitive charge-coupled device cameras, can be non-invasively detected in live animals. Currently, the most common luminescence endpoint is firefly luciferase, which, in the presence of O(2) and ATP, catalyses the cleavage of the substrate luciferin and results in the emission of a photon of light. In vivo implantation of tumour cells transfected with the luciferase gene allows sequential monitoring of tumour growth within the viscera by measuring these photon signals. Furthermore, tumour cell lines containing the luciferase gene transcribed from an inducible promoter offer opportunities to study molecular-target modulation without the need for ex vivo evaluations of serial tumour samples. In conjunction with this, transgenic mice bearing a luciferase reporter mechanism can be used to monitor the tumour microenvironment as well as to signal when transforming events occur. This technology has the potential to reshape the efficacy evaluations and drug-testing algorithms of the future.

Detection of genes for periplasmic nitrate reductase in nitrate respiring bacteria and in community DNA.

A nested PCR primed by four degenerate oligonucleotides was developed for the specific amplification of sequences from the napA gene encoding the periplasmic nitrate reductase. This approach was used to amplify fragments of the napA gene from 10 Pseudomonas species and one Moraxella sp., previously shown to be able to express the periplasmic nitrate reductase activity, from Rhodobacter capsulatus and from community DNA extracted from a fresh-water sediment. Amino acid sequences encoded by the napA fragments were compared to one another and to the corresponding regions of related enzymes. This comparison indicates that the amplification protocol is specific for its intended target. The napA sequences amplified from community DNA were tightly clustered, which may indicate a degree of homogeneity in the sediment community. All tested Gram-negative strains capable of aerobic nitrate respiration were found to have periplasmic nitrate reductase genes. However, some strains which have and express the genes are incapable of aerobic nitrate respiration. The PCR primers and amplification protocols described will be useful in future studies of nitrate respiring populations.

Isolation, characterization, and avenacin sensitivity of a diverse collection of cereal-root-colonizing fungi.

A total of 161 fungal isolates were obtained from the surface-sterilized roots of field-grown oat and wheat plants in order to investigate the nature of the root-colonizing fungi supported by these two cereals. Fungi were initially grouped according to their colony morphologies and then were further characterized by ribosomal DNA sequence analysis. The collection contained a wide range of ascomycetes and also some basidiomycete fungi. The fungi were subsequently assessed for their abilities to tolerate and degrade the antifungal oat root saponin, avenacin A-1. Nearly all the fungi obtained from oat roots were avenacin A-1 resistant, while both avenacin-sensitive and avenacin-resistant fungi were isolated from the roots of the non-saponin-producing cereal, wheat. The majority of the avenacin-resistant fungi were able to degrade avenacin A-1. These experiments suggest that avenacin A-1 is likely to influence the development of fungal communities within (and possibly also around) oat roots.

Dissimilatory Fe(III) reduction by Clostridium beijerinckii isolated from freshwater sediment using Fe(III) maltol enrichment.

A microorganism which reduces Fe(III) during the fermentation of glucose was isolated from freshwater sediment. The Fe(III) was supplied to enrichment cultures as a soluble complex with the bidentate ligand maltol (3-hydroxy-2-methyl-4-pyrone). Advantages that were afforded by the use of Fe(III)(maltol)3 over previously published methods included negation of the requirement for assays of Fe(II) formation. Because Fe(III)(maltol)3 has a characteristic deep red colour, Fe(III) reduction could be quantified spectrophotometrically by monitoring the disappearance of the complex in liquid cultures. Furthermore, Fe(III) reduction on agar plates containing the complex was apparent by zones of decolourisation around the bacterial colonies. 16S rRNA gene sequencing indicated the isolate to be a strain of Clostridium beijerinckii. Growth experiments were performed on the isolate in batch cultures with varying concentrations of Fe(III) citrate and 50 mM glucose. Increasing the level of Fe(III) citrate present was found to alter the fermentation balance, with less acidic products being formed. The presence of Fe(III) led to increases in the growth rate and growth yield, which were both approximately doubled when the supply of the cation reached 25 mM. A NAD(P)H-dependent Fe(III) reductase activity was localised to the bacterial membrane and found not to be sensitive to respiratory inhibitors. Taken together, these data suggest that dissimilatory Fe(III) reduction by the isolate provides a means of utilising the cation as an electron sink, thus facilitating pyridine nucleotide to be recycled during fermentative metabolism.

Hydroxylamine oxidation in heterotrophic nitrate-reducing soil bacteria and purification of a hydroxylamine-cytochrome c oxidoreductase from a Pseudomonas species.

Hydroxylamine oxidation was measured in four recently isolated heterotrophic nitrate-reducing bacteria belonging to the genera Pseudomonas, Moraxella, Arthrobacter and Aeromonas. A hydroxylamine-cytochrome c oxidoreductase activity was detected in periplasmic fractions of the Pseudomonas and Aeromonas spp. and in total soluble fractions of the Arthrobacter sp. A monomeric 19-kDa non-haem iron hydroxylamine-cytochrome c oxidoreductase was purified from the Pseudomonas species and shown to be similar to hydroxylamine-cytochrome c oxidoreductase of Paracoccus denitrificans.

Soil and sediment bacteria capable of aerobic nitrate respiration.

Several laboratory strains of gram-negative bacteria are known to be able to respire nitrate in the presence of oxygen, although the physiological advantage gained from this process is not entirely clear. The contribution that aerobic nitrate respiration makes to the environmental nitrogen cycle has not been studied. As a first step in addressing this question, a strategy which allows for the isolation of organisms capable of reducing nitrate to nitrite following aerobic growth has been developed. Twenty-nine such strains have been isolated from three soils and a freshwater sediment and shown to comprise members of three genera (Pseudomonas, Aeromonas, and Moraxella). All of these strains expressed a nitrate reductase with an active site located in the periplasmic compartment. Twenty-two of the strains showed significant rates of nitrate respiration in the presence of oxygen when assayed with physiological electron donors. Also isolated was one member of the gram-positive genus Arthrobacter, which was likewise able to respire nitrate in the presence of oxygen but appeared to express a different type of nitrate reductase. In the four environments studied, culturable bacteria capable of aerobic nitrate respiration were isolated in significant numbers (10(4) to 10(7) per g of soil or sediment) and in three cases were as abundant as, or more abundant than, culturable bacteria capable of denitrification. Thus, it seems likely that the corespiration of nitrate and oxygen may indeed make a significant contribution to the flux of nitrate to nitrite in the environment.

Isolation and characterisation of a strain of Pseudomonas putida that can express a periplasmic nitrate reductase.

A strain of Pseudomonas putida that can express a nitrate reductase that is located in the periplasmic compartment was isolated from freshwater. The enzyme was active in vivo during arginine fermentation and at the onset of oxygen limitation in batch cultures. The activity of the enzyme increased the yield of bacteria following fermentative growth under anoxic conditions with arginine, but nitrate reduction did not support growth on non-fermentable carbon substrates under anoxic conditions. Cells expressing the periplasmic nitrate reductase were capable of reducing nitrate in the presence of oxygen. Nitrate reduction under oxic conditions was clearly coupled to a respiratory electron transport chain because: (1) the process was sensitive to the respiratory inhibitors rote-none and 2-n-heptyl-4-hydroxyquinoline N-oxide, and (2) membrane-bound and periplasmic cytochromes were involved. This is the first report of the presence of a periplasmic nitrate reductase in a member of the gamma proteobacteria.

Suppression of in vivo clearance of N-nitrosodimethylamine in mice by cotreatment with ethanol.

Oral cotreatment of mice with ethanol results in increased tumors in extrahepatic organs caused by some nitrosamines. This action, attributed in part to inhibition of hepatic first-pass carcinogen metabolism by ethanol, has possible relevance to the enhancing effect of alcoholic beverage consumption on human cancer risk. In this study, the effects of ethanol on clearance of N-nitrosodimethylamine (NDMA) were quantified in Swiss female and strain A male mice. In Swiss mice, a 1.6 g/kg ig ethanol dose preceding 1 or 5 mg/kg iv NDMA resulted in 20- to 30-fold increases in area-under-the-blood-concentration-vs.-time curves, mean residence times, and clearance half-times, and similar decreases in clearance. For a 0.5 mg/kg ig NDMA dose, the pharmacokinetic parameters were altered 30-fold and 450-fold by simultaneous ethanol doses of 0.08 and 0.8 g/kg, respectively. With 5 mg NDMA/kg ig, 0.4, 0.8, and 1.6 g/kg ethanol resulted in 6-, 10-, and 20-fold changes in clearance parameters. Comparison of the data with results obtained previously with patas monkeys indicated comparable effects of ethanol on tissue exposure to NDMA in the two species, confirming potential human applicability. In experiments with strain A mice, NDMA concentrations were also monitored in lung and liver. NDMA amounts in lung paralleled those in blood, and were more than sufficient to account for the previously reported increases in DNA adducts and tumors in lungs of similarly treated strain A mice.

Hypothesis: dietary management may improve survival from nutritionally linked cancers based on analysis of representative cases.

A limited number of case histories was analyzed and verified to examine the effect of a very low fat, moderately high fiber, and moderately reduced calorie diet on the survival and quality of life of patients with primary cancer of the pancreas, metastatic stage D2 prostate cancer, and other nutritionally linked cancers. The retrospective study of pancreatic cancer patients disclosed that 1-year survival was higher among those who modified their diets than in those for whom there was no evidence as to diet alteration. For patients with metastatic prostate cancer (stage D2), a case control study demonstrated a statistical association of dietary modification with longer survival and improved quality of life. A retrospective study utilizing questionnaires supported such dietary modifications as a useful tool in the management of nutritionally linked cancers.

Synthesis and antimuscarinic activity of some 1-cycloalkyl-1-hydroxy-1-phenyl-3-(4-substituted piperazinyl)-2-propanones and related compounds.

A new class of substituted 1-phenyl-3-piperazinyl-2-propanones with antimuscarinic activity is reported. As part of a structure-activity relationship study of this class, various structural modifications, particularly ones involving substitution of position 1 and the terminal piperazine nitrogen, were investigated. The objective of this study was to derive new antimuscarinic agents with potential utility in treating urinary incontinence associated with bladder muscle instability. These compounds were examined for M1, M2, and M3 muscarinic receptor selectivity in isolated tissue assays and for in vivo effects on urinary bladder contraction, mydriasis, and salivation in guinea pigs. Potency and selectivity in these assays were influenced most notably by the nature of the substituent group on the terminal nitrogen of the piperazine moiety. Benzyl substitution was particularly advantageous in producing compounds with functional M3 receptor (smooth muscle) and bladder selectivity; it provided several candidates for clinical study. In vivo, 3-(4-benzyl-piperazinyl)-1-cyclobutyl-1-hydroxy-1-phenyl-2-propanone (24) demonstrated 11- and 37-fold separations in its effect on bladder function versus mydriatic and salivation responses, respectively. The corresponding 2-chlorobenzyl derivative 25 was more than 178-fold selective for M3 versus M1 and M2 muscarinic receptors. 3-(4-Benzylpiperazinyl)-1,1-diphenyl-1-hydroxy-2-propanone (51) was 18-fold selective for M3 versus M1 and 242-fold selective for M3 versus M2 receptors. It was also selective in guinea pigs, where it displayed 20- and 41-fold separations between bladder function and effect on mydriasis and salivation, respectively. In general, the results of this study are consistent with the proposition that the described piperazinylpropanones interact with muscarcinic receptors in a hydrogen-bonded form that presents a conformation similar to that apparently adopted by classical antimuscarinic agents.

Enhancement of tumorigenesis by N-nitrosodiethylamine, N-nitrosopyrrolidine and N6-(methylnitroso)-adenosine by ethanol.

Inclusion of 10% ethanol with 6.8 ppm N-nitrosodiethylamine in the drinking water of strain A male mice resulted in a 4-fold enhancement of multiplicity of lung tumors and a 16-fold increase in incidence of fore-stomach tumors, compared with carcinogen alone. Given with 40 ppm N-nitrosopyrrolidine, ethanol caused a 5.5-fold increase in lung tumor multiplicity. The inclusion of 15% ethanol with N6-(methylnitroso)adenosine, given orally to Swiss female mice, led to reduced body weights and shortened survival time related to hemangiosarcoma occurrence or increased incidence of thymic lymphoma, depending on dose of carcinogen. The data provide additional support for the proposal that co-administered ethanol increases the tumorigenicity of nitrosamines by blocking hepatic first-pass clearance.

Characterization of ethanol's enhancement of tumorigenesis by N-nitrosodimethylamine in mice.

The concentration-, time- and route-dependent effects of ethanol co-administration on tumorigenesis by N-nitrosodimethylamine (NDMA) were characterized in strain A male mice. With drinking-water administration, 1% ethanol was as effective as 5 or 10% in effecting a 4-fold enhancement of lung tumorigenesis by 5 p.p.m. NDMA. In a study of cumulative effects over time, 10% ethanol given with 1 p.p.m. NDMA resulted in a progressive increase in lung tumors from 16 to 72 weeks. In addition, at 72 weeks, the ethanol co-treatment resulted in a significant increase in kidney adenomas and possibly in vascular tumors of liver. A single i.g. dose of 5 mg/kg NDMA was significantly tumorigenic for lung, and the effect was dose-dependently increased by inclusion of ethanol, for up to a 9-fold enhancement with 20% ethanol. When 10% ethanol was given in the drinking water while NDMA was administered as 20 1 mg/kg doses by other routes--i.g., i.p., s.c. or i.v.--the ethanol treatment was without effect on lung tumor numbers. Collectively, the results provide strong support for inhibition of hepatic first-pass clearance of NDMA by cytochrome P450 2E1 as the mechanism of ethanol's effect, and suggest that several other possible mechanisms are unlikely. They also illustrate that a moderate dose of ethanol cumulatively increases tumor risk from a low dose of NDMA given over most of the lifetime of the animal.

Effects of a combination of evening primrose oil (gamma linolenic acid) and fish oil (eicosapentaenoic + docahexaenoic acid) versus magnesium, and versus placebo in preventing pre-eclampsia.

In a placebo controlled, partially double-blinded, clinical trial, a combination of evening primrose oil and fish oil was compared to Magnesium Oxide, and to a Placebo in preventing Pre-Eclampsia of Pregnancy. All were given as nutritional supplements for six months to a group of primiparous and multiparous pregnant women. Some of these women had personal or family histories of hypertension (21%). Only those patients who received prenatal care at the Central Maternity Hospital for Luanda were included in the study. Compared to the Placebo group (29%), the group receiving the mixture of evening primrose oil and fish oil containing Gamma-linolenic acid (GLA), Eicosapentaenoic acid (EPA), and Docosahexaenoic acid (DHA) had a significantly lower incidence of edema (13%, p = 0.004). The group receiving Magnesium Oxide had statistically significant fewer subjects who developed hypertension of pregnancy. There were 3 cases of eclampsia, all in the Placebo group.

Probing the bradykinin receptor: mapping the geometric topography using ethers of hydroxyproline in novel peptides.

Five decapeptides were prepared, each having the generic primary sequence D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-X7 -Y8-Arg9. A C-terminal beta-turn was anticipated when X was an alkyl ether of D-4-hydroxyproline in either the cis or trans geometric state and Y was either a Tic or Oic residue. Whereas cis ethers have only very weak receptor affinities, the trans ethers are significantly more potent in binding to guinea pig smooth muscle having Ki values as low as 0.16 nM. Notably, these peptides do not contain a D-aromatic amino acid at position 7 of the primary sequence.

Analogues of oxybutynin. Synthesis and antimuscarinic and bladder activity of some substituted 7-amino-1-hydroxy-5-heptyn-2-ones and related compounds.

Oxybutynin chloride [4-(diethylamino)-2-butynyl alpha-cyclohexyl-alpha-hydroxybenzeneacetate hydrochloride, Ditropan] is widely used for the relief of symptoms in neurogenic bladder. This is a result of its combined anticholinergic, antispasmodic, and local anesthetic activities. In a study directed toward development of agents possessing the beneficial properties of oxybutynin, but having a longer duration of action, a series of metabolically more stable keto analogues of the parent ester, i.e. substituted 7-amino-1-hydroxy-5-heptyn-2-ones along with some analogues and derivatives, was prepared and evaluated for in vitro and in vivo antimuscarinic action in guinea pig preparations. Several members of the series were potent antimuscarinics having a longer duration of activity than that of oxybutynin in a guinea pig cystometrogram model. On the basis of its in vitro and in vivo antimuscarinic activity, coupled with a 5-fold greater duration of action than that of oxybutynin, 1-cyclobutyl-7-(dimethylamino)-1-hydroxy-1-phenyl-5-heptyn-2-one (14b) was selected for clinical evaluation.