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Biomedical subjects

D L Kirkpatrick

Publications and source records attributed to D L Kirkpatrick.

At least 37 records · Page 2Linked to original sources
PubMed1994

Disulfide cytotoxicity under hypoxia.

The cytotoxicity of the disulfide n-butyl 2-imidazolyl disulfide (III-2) was determined to be the result of a disruption in the cellular redox state and inhibition of critical membrane enzymes. These events cause perturbations in Ca2+ homeostasis, which may affect the cell signalling machinery and cause the activation of catabolic enzymes. Exposure of EMT6 cells to III-2 resulted in depletion of nonprotein and protein thiols. Under hypoxic conditions, the depletion of reduced glutathione was less than that measured when cells were treated in air, whereas following an exposure to 500 microM III-2 for 2 h the enzymes glutathione S-transferase and glutathione reductase were inhibited to a greater extent under hypoxia. Ca2+ homeostasis was disrupted with an initial shift from the mitochondrial to the cytoplasmic pool. The inhibition of plasma membrane Ca(2+)-ATPase resulted in accumulation of Ca2+ in the cytoplasm. At higher concentrations, further disruption was seen as a net loss of Ca2+ of the cytoplasmic excess with no change in the mitochondrial levels, resulting in lower total cellular Ca2+. Neither the inhibition of Ca(2+)-ATPase nor the disruption of Ca2+ homeostasis were different under hypoxic vs. oxic conditions. Due to these observations, HL60 cells were used to measure whether III-2 stimulated apoptosis. Morphologic changes and DNA laddering were observed following exposure to the disulfide, with lower concentrations required to stimulate the cellular changes under hypoxia. These events may be the result of the disruption in Ca2+ homeostasis due to thiolation or alteration in redox status of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Reversible inhibition of human thioredoxin reductase activity by cytotoxic alkyl 2-imidazolyl disulfide analogues.

The thioredoxin/thioredoxin reductase system is important for several aspects of the regulation of cellular proliferation by both intracellular and extracellular mechanisms. The effects of n-butyl 2-imidazolyl disulfide (III-2), 1-methylpropyl 2-imidazolyl disulfide (IV-2), and n-decyl 2-imidazolyl disulfide (VII-2) on purified human placental thioredoxin reductase activity were examined. The analogues were competitive inhibitors with DTNB for reduction by thioredoxin reductase, with Ki values for III-2, IV-2, and VII-2 being 3.3, 13.0, and 8.6 microM, respectively. The inhibition was noncompetitive with reduced nicotinamide adenine dinucleotide phosphate (NADPH). None of the analogues was a suicide substrate inhibitor of the flavoenzyme. III-2 and VII-2 were metabolized by thioredoxin reductase at about half the rate of DTNB, whereas IV-2 was not detectably metabolized. The second order rate constants for the reactions of III-2 and IV-2 with reduced GSH were 931 and 91 M-1 s-1, respectively. The lower reactivity of IV-2 with reduced GSH and the lack of the analogue's metabolism by thioredoxin reductase may be due to the more sterically hindered structure of this analogue. The 50% inhibitory concentrations (IC50 values) for the inhibition of serum-dependent cellular proliferation of Swiss 3T3 murine fibroblasts by III-2, IV-2, and VII-2 were 2.0, 3.5, and 4.0 microM, respectively. IV-2 was considerably more potent as an inhibitor of the thioredoxin-dependent cellular proliferation of Swiss 3T3 fibroblasts, showing an IC50 value of 60 nM. Thus, inhibition of cellular proliferation by alkyl 2-imidazolyl disulfide analogues may involve interaction with thioredoxin, thioredoxin reductase, or an alternative target that is redox-regulated by thioredoxin.

Animals↗
PubMed1992

Synthesis and cytotoxic evaluation of some 6-arylidene-2-(alpha-hydroxyamino-alpha-arylmethyl)cyclohexanone oximes and related compounds.

Reaction of 2,6-bis-(phenylmethylene)cyclohexanone (1) with a 4-molar excess of hydroxylamine hydrochloride and sodium acetate to produce the corresponding oxime 2 gave rise to 2-(alpha-hydroxyamino-alpha-phenylmethyl)-6-phenylmethylenecyclohexan one oxime (5a), whose structure was deduced from high-resolution proton nuclear magnetic resonance spectroscopy and confirmed by X-ray analysis. Compound 2 was eventually prepared from 1 with hydroxylamine per se and not with a mixture of hydroxylamine hydrochloride and sodium acetate. Ten analogues of 5a, namely 5b-5k, were prepared and evaluated for cytotoxicity. Six of the 11 compounds in series 5, as well as 1, showed activity in the 240-950 microM range against murine mammary EMT6 cells. Series 5 was also examined for cytotoxicity in an in vitro screen conducted by the National Cancer Institute with approximately 54 cell lines, and four compounds demonstrated selective toxicity toward various groups of tumors.

Animals↗
PubMed1992

Modification of the DiSC assay by the incorporation of monoclonal antibody staining.

The use of immunohistochemical staining with the DiSC assay is described. This modified staining procedure allows differentiation of viable human tumor cells from normal cells in biopsy preparations, facilitating the evaluation of slides from chemosensitivity assays. Samples from ALL and CLL patients were processed using both the DiSC and modified MoAb-DiSC procedure. The predictive results were compared and found to be identical. This procedure is advantageous in determining the chemosensitivity of tumor cells in a highly heterogeneous biopsy specimen.

Antibodies, Monoclonal↗
PubMed1992

Effect of a hypoxic tumor cell cytotoxic disulfide on the membrane and DNA of tumor cells in culture.

A disulfide, n-butyl 2-imidazolyl disulfide (III-2), recently reported to be more toxic to EMT6 tumor cells under hypoxia has also been shown to be preferentially toxic to KHT/iv cells under hypoxia with an IC90 value 3-fold lower versus that measured in air. The IC90 values for both cell lines were markedly affected when the pHe was decreased. Uptake studies revealed that the disulfide is metabolized at the cell membrane with only one of the metabolites, n-butanethiol, being taken up into the cell. There were no differences in the amount of uptake under oxia or hypoxia. Investigation of potential membrane damage revealed that III-2 was a mixed inhibitor of the enzyme Na+,K(+)-ATPase, from isolated plasma membrane. DNA damage, when examined using plasmid DNA in the absence or presence of glutathione, was absent. Minor single-strand breaks to EMT6 DNA could only be observed following exposure to III-2 at the highest concentrations tested, with no differences observed between treatments in air or under hypoxia.

Animals↗
PubMed1990

Chemosensitivity testing of fresh human leukemia cells using both a dye exclusion assay and a tetrazolium dye (MTT) assay.

Chemosensitivity testing using a DEA was performed on 91 leukemia samples from 70 patients with leukemia. In 55 patients, comparison of in-vitro results with actual response of the patient was made. There was good correlation in 82% of the cases, the results being highly significant p less than 0.001. The MTT assay was also adapted for the chemosensitivity testing and the usefulness of this assay was evaluated by comparing the results to the DEA for 24 of the samples. Survival results produced by the two assays were very similar. The MTT procedure was also found to be equally effective as the DEA as a predictive assay of chemosensitivity.

Antineoplastic Agents↗
PubMed1990

Evaluation of the cytotoxicity of some Mannich bases of acetophenone against the EMT6 tumour.

1-Phenyl-3-(4-morpholinyl)propan-1-one hydrochloride 1 and the related piperidino derivative 2 were shown to have activity against the EMT6 tumour in vitro. Modification of the structures of these compounds by introducing a glucosyloxy group onto the aryl rings lowered cytotoxicity but conversion of 1 and 2 into the corresponding bis-Mannich bases increased bioactivity considerably.

Acetophenones↗
PubMed1990

Cytotoxicity of Mannich bases of alpha-arylidene-beta-ketoesters and related compounds against EMT6 mammary carcinoma cells.

A number of Mannich bases 2 derived from alpha-arylidene-beta-ketoesters, some corresponding deaminated products 3, and a thiol adduct 5 were prepared. High resolution 1H NMR spectroscopy revealed that, in solution, most of the bases 2 existed principally in acyclic forms, but that all members of this series underwent some intramolecular cyclization. The compounds 2, 3 and 5 possessed activity against EMT6 mammary carcinoma cells. The Mannich bases 2a-e had the highest cytotoxicity. Topliss analysis of these compounds revealed an E4 parameter dependency, in which intramolecular cyclization was minimal. The Mannich base 2f--which existed principally in the cyclic forms 6 in deuterium oxide, the deamination products, and a thiol adduct had approximately one-sixth of the activity of 2a-e.

Animals↗
PubMed1989

Kinetic study of the interaction of glutathione with four antitumor disulfides: possible mechanism for cellular glutathione depletion.

The reactions between the cellular tripeptide, glutathione (GSH) and four disulfide derivatives of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) (compounds 1-4) were studied kinetically. The decyl and phenyl derivatives of 6-MP and 6-TG were reacted with GSH in phosphate buffer (pH 7.4 or 6.0) at 25.0 degrees C and were monitored spectrophotometrically by observing the release of 6-MP and 6-TG. Second order kinetics were observed, with rate constants of 142, 564, 4174 and 429 M-1 s-1 being measured for compounds 1-4, respectively. When the reactions were carried out in the presence of GSH-S-transferase the rates were enhanced 1.3-5.4 times those observed in the absence of enzyme. Products of the reactions were isolated by chromatography and tentatively identified by TLC or fast atom bombardment mass spectrometry. It was observed that GSH reacted with each disulfide in a 1:1 manner, forming a mixed disulfide between GSH and decanethiol or thiophenol while releasing 6-MP or 6-TG. It was concluded that the reported depletion of GSH from EMT6 cells after exposure to these disulfides could be due to their reaction with GSH, and the formation of the mixed disulfides.

Antineoplastic Agents↗
PubMed1989

Attempted modulation of disulfide antitumor activity in Balb/c mice through glutathione depletion.

We investigated the effects of buthionine sulfoximine (BSO)-mediated glutathione (GSH) depletion on the antitumor activity in Balb/c mice produced by four disulfide derivatives of 6-TG and 6-MP. Initial studies indicated that 14 h after BSO (5 mmol/kg) injections, tumor GSH levels were maximally depleted, while normal tissue GSH levels had returned to near control levels. Tumor growth delays and growth rates were compared for groups of animals receiving disulfides I-IV with and without BSO administration 14 h previous. Treatments with BSO alone produced no delay or growth rate differences from the control. Compounds II or III administered in the presence and absence of BSO also produced no delay or growth rate differences from control. Compound I (10 mg/kg) alone showed a delay of 5.2 days and a growth rate significantly slower than that of control (p = 0.05). In combination with BSO the effects were not enhanced. Compound IV (50 mg/kg) also produced delays in 2 separate trials (3.1 and 4.8 days) and significantly slower growth rates on each occasion compared to the control (p = 0.05). The growth rates were not significantly lowered in the presence of BSO. Administration of two doses of IV, 4 days apart, produced a delay (4.9 days) similar to that seen with a single dose. It produced 2 cures and was also more toxic, causing 3 deaths. Two doses of IV in combination with BSO pretreatment had a greater delay (16.0 days) and a significantly longer growth rate (p = 0.05) than two doses of IV alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Evaluation of some azines of aminomethylacetophenones and related quaternary ammonium compounds versus the EMT6 tumour.

Azines derived from 3-dimethylamino-1-phenyl-1-propanone hydrochloride and two ring dimethylaminomethyl-acetophenones as well as the related quaternary ammonium iodides were synthesized. In phosphate buffer ph 7.4/37 degrees C 3-dimethylamino-1-phenyl-1-propanone azine dimethoiodide (2) formed 1-phenyl-3-(3-phenyl-2-pyrazolin-1-yl)-1-propanone. In the presence of a biomimetic thiol, 2-mercaptoethanol, compound 2 gave a bis S-alkylated product namely 3-(2-hydroxyethylthio)-1-phenyl-1-propanone azine. New products were not observed when two quaternary ammonium compounds derived from ring aminomethylacetophenone azines were examined under similar conditions. Six derivatives had moderate activity against the EMT6 tumour in vitro at concentrations of 250-500 mumol.1(-1) and greatest potency was noted with the ring dimethylaminomethylacetophenone azines and related quaternary ammonium compounds at these concentrations.

Acetophenones↗
PubMed1987

Modification of antitumor disulfide cytotoxicity by glutathione depletion in murine cells.

Decyl and phenyl disulfide derivatives of 6-mercaptopurine and 6-thioguanine (6-TG) were examined for antineoplastic activity under aerobic and hypoxic conditions toward EMT6 cells in culture. Although these derivatives did not display selective toxicity toward the hypoxic cells, they were significantly more toxic than 6-TC to this cell line at 500 microM after a 2-h exposure. In conjunction with this cytotoxicity, these agents were found to deplete the cellular glutathione (GSH) levels to varying degrees at this same concentration after a 1-h period. Therefore, the effect of modulating the cellular GSH on the cytotoxicity of these agents was investigated. When the GSH was depleted to less than 5 or 41% of control levels, the cytotoxicity exhibited by these agents was significantly increased while that of 6-TG remained unchanged. The cytotoxicity of these agents was similar to that of decanethiol and thiophenol, the thiol portion of the molecules, both under normal treatment conditions or after depletion of GSH. The lack of selective toxicity toward hypoxic cells was correlated to the finding that the disulfides were broken down to the parents, 6-mercaptopurine, and 6-TG, by cells under aerobic conditions. However, these studies demonstrate that manipulation of GSH levels might yield a therapeutic gain for these disulfide derivatives of antitumor agents.

Animals↗
PubMed1986

(o- and p-Nitrobenzyloxycarbonyl)-5-fluorouracil derivatives as potential conjugated bioreductive alkylating agents.

A series of (o- and p-nitrobenzyloxycarbonyl)-5-fluorouracil derivatives were synthesized by reacting o- or p-nitrobenzyl chloroformate with 5-fluorouracil in the presence of triethylamine in DMF or Me2SO. The reductive activation of these agents was hypothesized to generate a reactive methide and 5-fluorouracil, two components that are capable of synergistic interaction through complementary inhibition. Measurement of the surviving fractions of EMT6 tumor cells treated with these agents in culture under conditions of hypoxia and aerobiosis resulted in equal cell kill regardless of the state of oxygenation. One of the synthesized agents, 3-(p-nitrobenzyloxycarbonyl)-5-fluorouracil (4), appeared to be superior to 5-fluorouracil in prolonging the survival time of mice bearing intraperitoneal implants of the P388 leukemia and Sarcoma 180.

Animals↗
PubMed1986

Nitrobenzyl derivatives as bioreductive alkylating agents: evidence for the reductive formation of a reactive intermediate.

o- and p-nitrobenzyl chlorides and carbamates were chemically and electrochemically reduced in the presence and absence of the nucleophile morpholine; activation of these compounds by reduction was required to produce an intermediate capable of alkylation. The reduction products formed by the catalytic hydrogenation of each compound were examined by gas chromatography-mass spectrometry. In addition, the products generated by controlled-potential electrolysis were examined by ESR and NMR spectrometry. After a one-electron reduction, o- and p-nitrobenzyl chlorides were activated to the nitrobenzyl radicals, which subsequently dimerized to the dinitrobibenzyl derivatives or reacted with morpholine when present in the reaction medium to form the (nitrobenzyl)morpholine adducts. The nitrobenzyl carbamates were not activated after a one-electron reduction; however, the morpholine and the ether adducts of these agents were observed after catalytic hydrogenation. It was assumed that an intermediate or intermediates formed after the one-electron reduction product, or the full reduction product of the carbamates, were capable of alkylating various nucleophiles. Chemical reduction of the potential bioreductive alkylating agent (o-nitrobenzyl)-6-thioguanine produced (o-aminobenzyl)-6-thioguanine, indicating a lack of formation of a reactive electrophile by reduction. (o-, (m-, and (p-nitrobenzyl)-6-thioguanine analogues were also examined for cytotoxic activity toward EMT6 tumor cells under aerobic and hypoxic conditions. In agreement with the inability of (o-nitrobenzyl)-6-thioguanine to form a reactive species after chemical reduction, no decrease in the survival of neoplastic cells exposed to 10(-4) M drug occurred under either aerobic or hypoxic conditions.

Alkylating Agents↗
PubMed1985

Effects of soft contact lenses on contrast sensitivity.

This study attempts to resolve the conflicting reports relative to contrast sensitivity function (CSF) and soft contact lens wear. Contrast sensitivity was measured at six spatial frequencies for 19 subjects (38 eyes) when corrected with either spectacles or soft lenses. Measured amounts of residual astigmatism and/or sphere were corrected using a trial frame and lenses. Additionally, data were evaluated on more than one occasion in order to investigate the effect of time upon visual performance with the lenses. The results indicate a measurable decrease in contrast sensitivity for only the highest of the spatial frequencies tested (22.8 c/deg) when soft lenses were worn. For those eyes demonstrating a clinically significantly decrease in contrast sensitivity, responsibility appears to be shared by both the contact lens and the cornea. There were no significant changes in CSF over time.

Contact Lenses, Hydrophilic↗