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J Cummings

Publications and source records attributed to J Cummings.

At least 19 recordsLinked to original sources
PubMed1992

The consequences of doxorubicin quinone reduction in vivo in tumour tissue.

A clear role for quinone reduction in the mechanism of action of doxorubicin has still to be established. There are three possible outcomes of this form of doxorubicin metabolism: (1) drug free radical formation, redox cycling and generation of reactive oxygen species (ROS) resulting in lipid peroxidation and DNA damage; (2) covalent binding of reactive drug intermediates to DNA; and (3) formation of an inactive 7-deoxyaglycone metabolite. In this work, the occurrence of each of these pathways has been studied in vivo in a subcutaneously growing rat mammary carcinoma (Sp 107). Doxorubicin was administered by direct intratumoural injection either as the free drug or incorporated in albumin microspheres (10-40 microns diameter). There was no evidence of an increase in lipid peroxidation over background after either treatment at any time point studied. In fact, doxorubicin administration resulted in a statistically significant reduction in lipid peroxidation at the later time points studied compared to control (no drug treatment), e.g. 24 hr: control, 21.7 +/- 2.8 SD nmol malondialdehyde/g tissue; free doxorubicin (70 micrograms drug), 14.5 +/- 4.0 SD nmol/g (P < 0.01 Student's t-test) and doxorubicin microspheres (70 micrograms drug), 17.4 +/- 1.1 nmol/g (P < 0.05). Covalent binding to DNA was measured by a 32P-post-labelling technique. Low levels of four putative drug-DNA adducts were detected; however, there were no qualitative or quantitative differences in profiles between free drug and microspheres. High 7-deoxyaglycone metabolite concentrations comparable to the parent drug itself were detected after administration of microspheres (3.0 micrograms/g +/- 1.7 SD at 24 hr and 3.1 micrograms/g +/- 1.1 SD at 48 hr). In contrast, these metabolites were present at levels close to the limit of detection of our HPLC assay after free drug (0.04 microgram/g +/- 0.03 SD at 24 hr and 0.02 microgram/g +/- 0.03 SD at 48 hr). Thus, 7-deoxyaglycone metabolite formation can occur in tumour tissue (indicating active drug quinone reduction) without concomitant increases in the level of lipid peroxidation or the levels of drug-DNA adducts. In conclusion, the main biological consequence of doxorubicin quinone reduction in vivo in tumour tissue would appear to be drug inactivation to a 7-deoxyaglycone metabolite rather than drug activation to DNA reactive species or ROS.

Albumins↗
PubMed1992

The enzymology of doxorubicin quinone reduction in tumour tissue.

We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.

Animals↗
PubMed1992

Studies on the molecular pharmacology of GR63178A. A novel pentacyclic pyrolloquinone anticancer drug.

GR63178A (NSC D611615) is the second pentacyclic pyrolloquinone to be evaluated clinically as an anticancer drug. Its mechanism of action is unknown but may be related either to its quinone group or planar ring system. In this report we have investigated the ability of GR63178A to bind non-covalently to DNA, inhibit topoisomerase II and undergo reduction to reactive free radical species. Using two DNA duplexes, a 12-mer oligonucleotide which is a preferred sequence for minor groove binders and a hexamer which is a preferred sequence for intercalators, no evidence of significant binding with GR63178A was found. Neither GR63178A nor GR54374X (its 9-hydroxy metabolite) inhibited purified human topoisomerase II in a decatenation assay. Free radical chemistry was studied by both pulse radiolysis and ESR spectroscopy as well as by in vitro drug incubations with NADPH-fortified rat liver microsomes and purified cytochrome P450 reductase. The one-electron reduction potential of GR63178A was -207 mV +/- 10 which is much more positive than other quinone-containing anticancer drugs such as doxorubicin, mitomycin C and mitozantrone. GR63178A underwent enzyme-catalysed quinone reduction more readily than doxorubicin but produced significantly fewer reactive oxygen species. No evidence was detected of drug-induced, radical-mediated DNA damage in vitro using pBR322 plasmid DNA. Disproportionation of the GR63178A semi-quinone free radical proceeded with a rate constant of 1 x 10(9) M-1 sec-1 under anaerobic conditions, one order of magnitude faster than doxorubicin. The preferential disproportionation of the semi-quinone may explain our inability to detect a free radical signal by ESR. The hydroquinone of GR63178A was stable and exhibited strong visible absorption with a bathochromic shift of 120 nm over the parent drug. These unusual properties may be due to the hydroquinone undergoing a form of keto-enol tautomerization. Thus, GR63178A free radical formation does not appear to result in significant drug activation. In conclusion, GR63178A is unlikely to mediate its antitumour activity by DNA binding, topoisomerase II inhibition or free radical formation in direct contrast to similar anthracycline- and anthraquinone-based anticancer drugs.

Animals↗
PubMed1992

Rapid and selective isolation of radiolabelled inositol phosphates from cancer cells using solid-phase extraction.

A method is described for rapid and selective determination of radiolabelled inositol phosphates in cancer cells using solid-phase extraction with Bond Elut strong anion-exchange minicolumns. The inositol phosphates IP1, IP2 and IP3 are selectively eluted with 0.05, 0.3 and 0.8 M ammonium formate-0.1 M formic acid, respectively. Cancer cells are extracted with 10% perchloric acid which is then neutralised prior to loading samples on to the minicolumns. Recovery is 54.1, 66.6 and 61.3% for IP1, IP2 and IP3 with between-day coefficients of variation of 7.6, 6.8 and 1.9%, respectively. When the method was applied to cancer cells high-performance liquid chromatographic analyses confirmed both the identity of the IP1, IP2 and IP3 fractions and showed that there was no detectable cross contamination of these inositol phosphates with each other.

Chromatography, High Pressure Liquid↗
PubMed1992

A phase I and pharmacology study of GR63178A, a water-soluble analogue of mitoquidone.

GR63178A is a water-soluble analogue of mitoquidone, a pentacyclic pyrroloquinone. This group of drugs exhibit a novel structure and activity against several murine solid tumours and xenografts. In the present phase I study the toxicity and pharmacokinetics of GR63178A given on 5 consecutive days of a 21-day cycle were examined. A total of 24 patients presenting with a wide range of tumours were treated at 5 doses escalated to reach the maximal tolerated dose (MTD). Linear pharmacokinetics was documented over the dose range studied, and there was no difference in parent drug handling between day 1 and day 4 of dosing. A number of metabolites were detected. The toxicity profile was unusual in that pain occurred in 20/24 patients, most often at the site of known disease. This was the dose-limiting toxicity. Other side effects included nausea and vomiting (23/24), phlebitis at the infusion site (6/24) and headache (7/24). No treatment response was seen in this study. The MTD was demonstrated to be 160 mg/m2 daily (total, 800 mg/m2 per treatment cycle). The drug has now entered phase II trials at 120 mg/m2 daily x 5, repeated every 21 days.

Adult↗
PubMed1992

Doxorubicin-loaded casein microspheres: protein nature of drug incorporation.

We have studied incorporation of [14C]doxorubicin within protease-sensitive casein microspheres both by 14C-activity, measuring total drug, and HPLC, measuring free drug only. It was found that total drug content (27.7 micrograms mg-1) exceeded free drug content (3.2 micrograms mg-1) suggesting that the major portion of doxorubicin was incorporated via a covalent linkage to matrix protein. In-vivo drug disposition and activity studies suggested that this fraction of doxorubicin was the major species within tumour tissue (total vs free: 5 min, 14.3 micrograms g-1 vs 0.7 micrograms g-1; 24 h, 11.7 micrograms g-1 vs 1.1 micrograms g-1; 48 h, 11.2 micrograms g-1 vs 1.2 micrograms g-1; 72 h, 10.0 micrograms g-1 vs 0.8 micrograms g-1), did not exhibit a 'burst' effect, was slowly cleared (30% loss over 3 days), and was equiactive (growth delay = 12 days) compared with drug in solution (growth delay = 10 days). This work clearly implicates in-vivo microsphere matrix biodegradation in drug release and subsequent disposition and activity.

Animals↗
PubMed1992

A randomized trial of the cost effectiveness of VA hospital-based home care for the terminally ill.

All admissions to a 1,100-bed Department of Veterans Affairs (VA) hospital were screened to identify 171 terminally ill patients with informal caregivers who were then randomly assigned to VA hospital-based team home care (HBHC, N = 85) or customary care (N = 86). Patient functioning, and patient and caregiver morale and satisfaction with care were measured at baseline, one month, and six months. Health services utilization was monitored over the six-month study period and converted to cost. Findings included no differences in patient survival, activities of daily living (ADL), cognitive functioning, or morale, but a significant increase in patient (p = .02) and caregiver (p = .005) satisfaction with care at one month. A substitution effect of HBHC was seen. Those in the experimental group used 5.9 fewer VA hospital days (p = .03), resulting in a $1,639 or 47 percent per capita saving in VA hospital costs (p = .02). As a result, total per capita health care costs, including HBHC, were $769 or 18 percent (n.s.) lower in the HBHC sample, indicating that expansion of VA HBHC to serve terminally ill veterans would increase satisfaction with care at no additional cost.

Activities of Daily Living↗
PubMed1991

The role of protein kinase C and the phosphatidylinositol cycle in multidrug resistance in human ovarian cancer cells.

The present study aimed to investigate the role of protein kinase C (PKC), the phosphatidylinositol pathway (PI) and cytosolic calcium in multidrug resistance (MDR) in human ovarian carcinoma cells. Binding of the phorbol ester 13,14-dibutyrate (PDBu) was 3-fold higher in resistant A2780AD versus sensitive A2780 cells indicating increased PKC activity. However, when inositol phosphate production (IP) was measured in quiescent cells similar total IP release was seen in both lines suggesting no difference in the basal turnover of PI. Non-specific stimulation of the PI pathway was achieved with the calcium ionophore A23187 which increased IP production in a time- and dose-dependent fashion in both cell lines but was significantly less effective in A2780AD. The PI pathway was investigated further using the agonists aluminium fluoride, serum and bombesin but these agents failed to elicit a response. The effect of a wide range of Adriamycin concentrations on the PI cycle and cell growth was also studied. Intracellular calcium was measured with the fluorescent dye fura-2-pentaacetoxymethylester (Fura-2). A23187 produced a rise in cytosolic calcium in A2780 and A2780AD but from a level 3-fold lower in the unstimulated resistant cell line. The dose responsiveness of this effect was greater but irreversible in A2780AD cells. Collectively these results imply that alterations in PI turnover appear not to be responsible for the differences in PDBu binding and calcium handling observed between A2780 and A2780AD and suggests only a minor role for the PI cycle in the maintenance of MDR in human ovarian cancer cell lines.

Calcimycin↗
PubMed1991

Determination of reactive nitrogen mustard anticancer drugs in plasma by high-performance liquid chromatography using derivatization.

A high-performance liquid chromatography (HPLC) method is described for the determination of reactive nitrogen mustard anticancer drugs in plasma after derivatization with diethyl-dithiocarbamic acid (DDTC). Three compounds were studied: two reactive species (mechlorethamine (HN2) and galactose 6-mustard (G-6-M] and a less reactive species (melphalan (L-PAM] included for validation experiments. Mass and NMR spectrometry confirmed that one molecule of DDTC reacts with each arm of the mustard, displacing a chlorine atom to form a stable disubstituted adduct. With the reactive mustards a 30-min incubation at 37 degrees C is recommended for greater than 90% derivatization efficiency. Gradient elution was employed to analyze all three compounds using the same conditions with a microBondapak C18 10-microns particle size column (30 cm by 3.8 mm i.d.). The retention time (tR) of HN2-DDTC2 was 13.1 min +/- 1.5% within day CV; tR of L-PAM was 7.6 min and L-PAM-DDTC2 was 14.6 min +/- 0.8% CV. G-6-M-DDTC2 yielded a double peak, tR = 10.7 min and 10.9 min +/- 2.9% CV. The limit of detection on column was 0.5 ng for HN2, 1 ng for L-PAM, and 5 ng for G-6-M. A solid-phase sample preparation technique using "Bond Elut" phenyl is described that extracts from plasma G-6-M-DDTC2 with greater than 74% efficiency and HN2-DDTC2 with greater than 90% efficiency. When the drugs were derivatized in plasma, recovery remained high for G-6-M (greater than 84%) but dropped to 50% for HN2.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗
PubMed1991

Covalent coupling of doxorubicin in protein microspheres is a major determinant of tumour drug disposition.

Doxorubicin is shown to be present in albumin microspheres (10-40 microns) in two forms: the native drug and a fraction of drug covalently coupled to the protein matrix probably via glutaraldehyde. Upon trypsin digestion the fraction covalently coupled is released and can be resolved from native doxorubicin by high performance liquid chromatography and quantitated either by using 14C-labelled doxorubicin or by measuring the absorption of the doxorubicin chromophore at 480 nm. Albumin microspheres contained 6.9 micrograms/mg protein covalently bound drug versus 11.1 micrograms/mg native drug when 1% glutaraldehyde was used in microsphere preparation. The covalently bound fraction increased significantly with 2% glutaraldehyde. Albumin/polyaspartic acid microspheres lacked a covalently bound fraction when prepared under the same conditions as pure albumin microspheres (35 micrograms/mg native drug, 1% glutaraldehyde) but transferrin microspheres contained similar amounts of bound and native albumin. In vivo, albumin microspheres altered the disposition of doxorubicin in a rat mammary carcinoma (Sp107) compared to albumin/polyaspartic acid microspheres by reducing the rate of parent drug elimination from the tumour and by reducing its biotransformation to 7-deoxyaglycone metabolites. These data indicate that covalent coupling is a key component in the way doxorubicin is handled in tumours after administration of protein microspheres.

Albumins↗
PubMed1991

Chromatographic characterisation of six human metabolites of the new anticancer drug GR63178A.

GR63178A is the second pentacyclic pyrroloquinone to enter clinical trials as an anticancer drug. We developed a reversed-phase, gradient-elution high-performance liquid chromatography (HPLC) method along with a Bond Elut C2 mini-column sample-preparation technique for the analysis of GR63178A, its 9-hydroxy-metabolite GR54374X and internal standard GR70440A in human plasma and urine. The limit of detection is 2 ng/ml for both GR63178A and GR54374X. Analysis of GR63178A is complicated by its light instability, whereby a number of chromatographically distinct, stable degradation products can form. These can be practically eliminated if clinical specimens are frozen immediately and all subsequent sample preparation is performed in a darkroom. Using this methodology, a total of six metabolites (including GR54374X) were detected in human plasma and urine specimens. The five new metabolites were characterised according to polarity (HPLC retention time), UV-visible absorption maxima and the effect of incubation with beta-glucuronidase and aryl-sulphatase. Application of this methodology to the analysis of GR63178A will aid in the development of this novel synthetic anticancer drug.

Antineoplastic Agents↗
PubMed1991

Phase I study of the anthrapyrazole biantrazole: clinical results and pharmacology.

In a phase I study the anthrapyrazole biantrazole (Warner-Lambert Company) was given to 41 patients with tumour refractory to existing therapy. The drug was given i.v. weekly for 3 weeks, with a 3-week interval between courses. At the 1st week a full pharmacokinetic study was performed, and at weeks 2 and 3, blood samples were taken at 1 and 6 h following treatment to check for drug accumulation. Biantrazole pharmacokinetics were linear with respect to the AUC (r = 0.924) over the full range of doses studied (4-36 mg/m2) but exhibited large inter-patient variations at each dose level. Elimination was triphasic, comprising two rapid early phases and a long terminal half-life (mean, 14.1 +/- 7.8 h). There was no evidence of drug accumulation over the 3-week treatment period. Approximately 12% of the parent drug was excreted unchanged in the urine together with two non-circulating, more water-soluble metabolites. Biantrazole was well tolerated but did cause moderate emesis at doses of greater than 18 mg/m2 and mild alopecia. The dose-limiting side effect was leucopenia, with no other major toxicity being observed. One patient developed biventricular failure that was not clearly related to biantrazole administration. On the present schedule, the recommended dose of biantrazole is 24 mg/m2. No response were seen in this patient population.

Adolescent↗
PubMed1991

Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase.

An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-microns DEAE anion-exchange resin capable of chromatographing nucleic acids greater than 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 M potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When [14C]doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 M sodium chloride at 50 degrees C for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single-stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA (34 pmol/100 micrograms), and the highest levels were recorded with oligonucleotides (243 pmol/100 micrograms). The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 microM [14C]doxorubicin (18.6 pmol/100 micrograms). Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques.

Animals↗