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P Workman

Publications and source records attributed to P Workman.

At least 181 records · Page 10Linked to original sources
PubMed1987

Misonidazole and benznidazole inhibit hydroxylation of CCNU by mouse liver microsomal cytochrome P-450 in vitro.

On the basis of promising experimental studies, the nitroimidazoles misonidazole (MISO) and benznidazole (BENZO) are under clinical investigation as chemosensitizers in combination with the chloroethylnitrosourea CCNU. We have shown previously that MISO and BENZO can alter the pharmacokinetics of CCNU leading to an improved therapeutic index in mice. Here we demonstrate using optical difference spectroscopy that MISO and BENZO are able to bind to cytochrome P-450 of mouse liver microsomes in vitro. Binding was type II in nature, indicating co-ordination of the free imidazole nitrogen with the heme moiety of cytochrome P-450. This results in an inhibition of CCNU hydroxylation by the hemoprotein. The kinetics of inhibition were of a mixed competitive-non-competitive type. At a CCNU concentration of 0.05 mM the concentrations causing 50% inhibition (I50) were 5.8 and 0.37 mM for MISO and BENZO respectively. At doses producing a similar improvement in therapeutic index in mice (2.5 mmoles/kg MISO and 0.3 mmoles/kg BENZO) the plasma and tissue concentrations achieved would inhibit CCNU hydroxylation by 30%. For BENZO, but not MISO, similar inhibition would also occur at concentrations which can be achieved safely in man.

Animals↗
PubMed1987

Nitroimidazole bioreductive metabolism. Quantitation and characterisation of mouse tissue benznidazole nitroreductases in vivo and in vitro.

We have investigated the nitroreduction of the 2-nitroimidazole benznidazole (BENZO) to its corresponding amine by murine normal tissues and tumours. In vivo concentrations of BENZO and its amine metabolite were measured by HPLC 3 hr after BENZO, 2.5 mmoles kg-1 i.p. This gave plasma and tissue BENZO concentrations of 96-160 micrograms ml-1 or g-1. Mouse plasma, KHT and RIF-1 tumour BENZO amine concentrations were very low (0.3-1.4 micrograms g-1); kidney and EMT6 tumours had intermediate levels; and liver contained very high amine levels (approximately 50 micrograms g-1). Three per cent of the BENZO dose was recovered as amine in the 24 hr urine, compared to 5% for the parent compound. Nitroreduction to the amine was demonstrated with liver and tumour preparations under N2 in vitro. The reaction was highly dependent on NADPH, and inhibited extensively in air. With liver microsomes and whole homogenates 2 and 3 moles respectively of BENZO were consumed per mole of amine formed. Inhibitor studies showed that NADPH: cytochrome P-450 (cytochrome c) reductase and cytochrome P-450 were both involved in BENZO reduction, predominantly at early and late reduction steps respectively. Aldehyde oxidase contributed to the cytosolic nitroreduction. Purified buttermilk xanthine oxidase also reduced BENZO to its amine under anaerobic conditions in vitro, but very inefficiently. The apparent Km and Vmax for BENZO amine production by whole liver homogenates were 0.148 mM and 1.45 nmole min-1 mg-1 protein respectively. Tumour homogenates were less active than liver; e.g. Vmax for the KHT tumour was 6-10-fold lower.

Aldehyde Oxidase↗
PubMed1987

Improved methodology for intracellular enzyme reaction and inhibition kinetics by flow cytometry.

Flow cytoenzymology is the determination of enzyme activities or concentrations in single intact cells. Using the flow cytometer built and designed in our laboratory and recent modifications to hardware and software, we have developed an improved dynamic flow cytoenzymological procedure for the assay of cellular enzyme kinetics. The reaction mixture is sampled continuously, and the computer clock incorporates time as a parameter for kinetic determinations. Conditions for cellular esterase analysis were optimized and the rates of hydrolysis of two fluorogenic substrates, fluorescein diacetate (FDA) and 4-methylumbelliferone acetate (MUA), by esterases in EMT6 mouse mammary tumor cells were studied. Reaction kinetics were characterized, and Km values of 19 and 72 microM were obtained for the hydrolysis of FDA and MUA respectively. The kinetics of the cellular efflux of fluorescein were investigated, and a half-life of 7.5 min obtained. Enzyme inhibition kinetics were investigated using the competitive substrates p-nitrophenyl acetate and phenyl acetate, and the carbamoylating agents physostigmine and n-butyl isocyanate. The latter was particularly potent with an I50 of 4.8 X 10(-5) M for FDA hydrolysis compared with 6.5 X 10(-3) M for physostigmine. The I50 of 8.8 X 10(-5) M for n-butyl isocyanate inhibition of MUA hydrolysis was similar to that obtained with FDA as substrate. By monitoring FDA and MUA reactions separately and simultaneously, we showed them to act as competitive substrates. A comparison of flow cytoenzymological and conventional spectrofluorimetric analysis was also made, and differences identified in some cases.

Animals↗
PubMed1987

The effects of whole body hyperthermia on the pharmacokinetics and toxicity of the basic 2-nitroimidazole radiosensitizer Ro 03-8799 in mice.

We have investigated the effects of 50 min whole-body hyperthermia (WBH; 15 min equilibration followed by 41 degrees C for 35 min) on the toxicity and pharmacokinetics of the radiosensitizer Ro 03-8799 in mice. WBH markedly reduced Ro 03-8799 LD50/7d from 779 to 259 micrograms g-1 (P less than 0.001). Pharmacokinetics were studied at 175 micrograms g-1 (approximately 0.6 WBH LD50/7d) with and without heat and 437 micrograms g-1 (approximately 0.6 control LD50/7d) without heat. WBH increased Ro 03-8799 plasma concentrations and prolonged its elimination t1/2 by 26% (P less than 0.01). Total plasma area under the curve (AUC0-infinity) was increased by 22%, but was still less than 50% of the unheated high-dose value. Ro 03-8799 concentrated 300-400% in tumour and brain relative to plasma. Absolute tumour and brain levels were unaltered by WBH, giving reduced tissue/plasma ratios. WBH greatly inhibited glomerular filtration (51Cr EDTA clearance) during heating, contributing to the increased plasma Ro 03-8799 concentrations. WBH increased peak plasma concentrations of the Ro 03-8799 N-oxide metabolite Ro 31-0313 by 61% and the beta-phase AUC of i.v. administered Ro 31-0313 by 36%. Since Ro 31-0313 levels were increased to a greater extent after Ro 03-8799 and WBH than Ro 31-0313 and WBH, WBH must both increase metabolite production and decrease its plasma clearance. WBH had no effect on Ro 31-0313 tumour concentrations or its exclusion from brain. These complex effects of WBH on Ro 03-8799 pharmacokinetics may contribute to the enhanced toxicity, possibly through hyperthermia-stimulated bioreductive drug activation, but do not wholly explain it.

Animals↗
PubMed1986

Estimating the kinetic parameters for enzymatic drug metabolism in the whole animal.

A method for estimating the Michaelis constant, Km, and the maximum reaction velocity, Vmax, for the enzymatic degradation of a parent compound to a metabolite in the intact animal is presented. The technique involves a mathematical analysis which has shown that under specific conditions the peak/plateau blood concentrations of metabolite are related to initial parent compound concentration by the Michaelis-Menten relationship. It has also been shown how these data can be analysed with the "direct linear plot" of Eisenthal and Cornish-Bowden (Biochem. J. 139, 715 (1974)) to yield the enzyme kinetic parameters.

Animals↗
PubMed1986

CB 1954 revisited. I. Disposition kinetics and metabolism.

Although it has been the subject of considerable interest for 15 years, originally as a cytotoxic agent and more recently as a radiosensitizer, there is very little pharmacokinetic information on CB 1954 (2,4-dinitro-5-aziridinylbenzamide). We have developed a rapid high-performance liquid chromatography assay for the drug and its metabolites and applied it to detailed examination of the pharmacokinetics of CB 1954 in mice and dogs. With IV administration a dose of 50 mg/kg gave peak blood concentrations of 100 micrograms/ml in mice, while 25 mg/kg gave peak plasma concentrations of 27 micrograms/ml in dogs. Peak concentrations were 3 to 5-fold lower for the IP route in mice and the oral route in dogs, and the bioavailabilities were 85% and 40%, respectively. Elimination t1/2 values were 1.4-2 h in mice and 2.5-4 h in dogs and were independent of route of administration. Plasma protein binding was 57% but tissue penetration in mice was generally good. Tumour: plasma ratios were 50%-90%, while brain: plasma ratios were lower, at 37%-50%. The parent drug and several metabolites were identified and quantified in mouse urine, the total recovery being 24%-29%, of which 16%-25% was parent drug. The metabolites were also found in the circulation and in tissues. No changes in pharmacokinetics were seen with repeated dosing in mice or with administration of the protective agent phenyl AIC. Phenobarbitone pretreatment produced a small reduction in elimination t1/2, mainly by accelerating aziridine ring removal. Allopurinol increased the blood levels of the 5-amino nitroreduction product. These studies provide a pharmacokinetic basis for interpreting the antitumour activity and toxicity of CB 1954, as well as for the development of new 'mixed-function' sensitizers.

Allopurinol↗
PubMed1986

CB 1954 revisited. II. Toxicity and antitumour activity.

We have assessed the antitumour activity of the nitrophenylaziridine CB 1954 in vitro and in vivo. For EMT6 mouse mammary tumour multicellular spheroids under hypoxic conditions in vitro, a 6-h exposure to 40 micrograms/ml reduced the surviving fraction to as low as 10(-3) and the growth delay was 5.4 days. Oxic cells were twofold less sensitive. Phenyl AIC protected oxic and hypoxic cells equally. Under oxic conditions minimal cell killing was seen with HT29 cells, either in multicellular spheroids or in monolayer; a 6-h exposure to 40 micrograms/ml gave a spheroid growth delay of 1.5-1.7 days. No growth delay was seen with single maximum tolerated doses of CB 1954 against HT29 grown as a xenograft in immunosuppressed mice. Only minimal growth delays of 1-2 days were seen with similar doses against the EMT6 tumour and the RIF-1 and KHT sarcomas in mice. Little activity was seen with maximum tolerated doses given once a day for 5 days against EMT6 and RIF-1. No chemosensitization was measurable with CCNU, cyclophosphamide or melphalan in the KHT tumour.

Animals↗
PubMed1986

Pharmacokinetic basis for the comparative antitumour activity and toxicity of chlorambucil, phenylacetic acid mustard and beta, beta-difluorochlorambucil (CB 7103) in mice.

This report describes the relationship between the pharmacokinetics, antitumour activity and toxicity of chlorambucil (CHL), phenylacetic acid mustard (PAAM) and beta, beta-difluorochlorambucil (beta-F2CHL) in mice. Pharmacokinetics were studied by HPLC, antitumour activity by a regrowth delay assay using the KHT murine sarcoma and toxicity by acute LD50. For both antitumour activity and acute toxicity the order of potency was: PAAM greater than CHL greater than beta-F2CHL. CHL and PAAM exhibited identical therapeutic indices, whereas that for beta-F2CHL was somewhat improved. CHL is metabolized by mitochondrial beta-oxidation to the 3,4-dehydro derivative (DeHCHL) and PAAM, and the latter is further metabolized to its monodechloroethylated derivative DeC-PAAM, presumably by hepatic microsomal enzymes. Administered PAAM gave only one metabolite, DeC-PAAM. Unexpectedly, despite beta, beta-disubstitution, beta-F2CHL was also beta-oxidized to give DeHCHL and PAAM, but at reduced rates. Further, metabolic switching was demonstrated with the appearance in large amount of 2 new, unidentified metabolites, which may be dechlorethylation products. The pharmacokinetics of administered CHL, PAAM and beta-F2CHL differ in that the plasma clearance was fastest for CHL, slowest for PAAM and intermediate for beta-F2CHL. For the metabolites, CHL produced peak plasma concentrations of DeHCHL and PAAM, respectively, 7-fold and 2-fold greater than those produced by beta-F2CHL. However, despite these differences, exposures to total bifunctional nitrogen mustards were similar following administration of the 3 drugs and therefore cannot account for their differential activity. In contrast, there was a good correlation between potency and PAAM exposure, which is highest after treatment with PAAM, intermediate after CHL and lowest after beta-F2CHL. In plasma, 3.2% of PAAM is present as nonprotein-bound free drug, compared to 1.3% for DeHCHL, 0.9% for CHL and 0.45% for beta-F2CHL. We propose the amount of free bifunctional nitrogen mustard, itself partly dependent on the extent of metabolism, to be of major importance for the in vivo potency of CHL analogues.

Animals↗
PubMed1986

Altered pharmacokinetics in the mechanism of chemosensitization: effects of nitroimidazoles and other chemical modifiers on the pharmacokinetics, antitumour activity and acute toxicity of selected nitrogen mustards.

We have studied the effect of misonidazole (MISO) on the antitumour activity, normal tissue toxicity and pharmacokinetics of four bifunctional nitrogen mustards: chlorambucil (CHL); phenylacetic acid mustard (PAAM), a metabolite of CHL; beta, beta-difluorochlorambucil (beta-F2CHL), an analogue which is metabolized less efficiently by the beta-oxidation pathway; and melphalan (MEL). MISO (2.5 mmol/kg) increased the response of the KHT tumour to CHL, PAAM and beta-F2CHL by dose-modifying factors (DMFs) of 1.55-1.85, 1.35-1.65 and 1.5-1.8, respectively. In contrast, the activity of MEL was not altered. However, with 5.0 mmol/kg MISO an enhanced response to MEL was observed (DMF = 1.35-1.55). Similarly, for CHL and PAAM, but not MEL, acute toxicity was also increased by 2.5 mmol/kg MISO. The increase in toxicity with CHL and PAAM was similar to the increase in antitumour activity, and their therapeutic indices were unchanged. Effective chemosensitizers were shown to be powerful inhibitors of drug clearance. Thus, potent chemosensitizers such as MISO, the lipophilic analogue benznidazole (BENZO), the microsomal enzyme inhibitor SKF 525A, and the parent heterocycle imidazole all reduced the plasma clearance of CHL and its metabolites and therefore increased drug exposure (AUC). Conversely, the hydrophilic MISO metabolite Ro 05-9963 was a poor chemosensitizer and produced only very weak pharmacokinetic effects. As the DMFs for chemosensitization agreed very well with those for increased AUC, it seems likely that pharmacokinetic changes are the major cause of the enhancement of tumour response to CHL. For MEL, chemosensitization also appears to be related to pharmacokinetic changes. MISO at a dose of 2.5 mmol/kg produced no change in MEL pharmacokinetics and no enhancement of tumour response, whereas 5.0 mmol/kg MISO was effective on both counts.

Animals↗
PubMed1986

Interaction of nitroimidazole sensitizers with drug metabolizing enzymes--spectral and kinetic studies.

We have investigated the effect of a range of 2-nitroimidazoles on CCNU metabolism, using an in vitro mouse liver microsomal preparation. CCNU is hydroxylated to at least 5 monohydroxylated metabolites. For the major metabolite, cis-4-hydroxy CCNU, values of Km and Vmax were 0.026 mM and 1.92 nmol/min/mg protein, respectively. MISO and other 2-nitroimidazoles inhibited the hydroxylation of CCNU in a dose-dependent manner and their potencies as inhibitors were governed by their lipophilicities. In order of increasing potency I50 values were 15.5, 6.4, 5.8, 1.4, 0.4, and 0.37 mM for SR 2508, Ro 03-8799, MISO, Ro 07-1902, Ro 07-1127, and BENZO, respectively. Chemosensitization potency correlated well with the extent of inhibition at achieved plasma concentrations in mice, suggesting a causal relationship between enzyme inhibition and chemosensitization. All the nitroimidazoles exhibited type II optical difference spectra with phenobarbitone-induced mouse liver microsomes. However, with increasing lipophilicity of the nitroimidazole both the wavelength at maximum absorbance (lambda max) and the isosbestic point of the type II spectrum were shifted to longer wavelengths, suggesting that a type I binding component may become more significant. Our previous work has shown that changes in CCNU pharmacokinetics contribute to chemosensitization by nitroimidazoles in mice, and that altered pharmacokinetics also occur in man. The present results provide strong evidence that the mechanism involves binding to liver microsomal cytochrome P-450, leading to inhibition of CCNU metabolism.

Animals↗
PubMed1986

A phase I study of the combination of two hypoxic cell radiosensitizers, Ro 03-8799 and SR-2508: toxicity and pharmacokinetics.

The hypoxic cell radiosensitizer Ro 03-8799 produces acute central nervous system toxicity which limits repeated doses of the drug to 0.75 g/m2, but peripheral neuropathy does not occur. SR-2508 causes no acute effects at doses greater than 3.0 g/m2, but causes peripheral neuropathy at cumulative doses of 30 g/m2. By combining maximum tolerated doses of each agent, it may be possible to increase efficacy, but not toxicity. Escalating single doses of Ro 03-8799 and SR-2508 were administered to 10 patients. The drugs were infused together in 50 ml of 0.9% saline over 10 min, beginning at 0.5 g/m2 of each agent, and proceeding to a fixed dose of 0.75 g/m2 Ro 03-8799 with 0.5, 1.0, 2.0, and 3.0 g/m2 SR-2508. Four patients experienced the expected acute syndrome related to Ro 03-8799, but the incidence was not increased by escalating doses of SR-2508, and no peripheral neuropathy was seen. Plasma and urine pharmacokinetic studies showed that no drug interaction occurred. Six patients have been given a 9-dose regime over a 3 week period, using 0.75 g/m2 Ro 03-8799 and escalating doses of 0.5, 1.0, and 1.5 g/m2 SR-2508. All exhibited the expected acute side effects related to Ro 03-8799, but with no increase at the higher doses of SR-2508. No other toxicity was seen. Plasma pharmacokinetics performed at the beginning and end of the schedule were similar. Biopsies were taken from six superficial tumors following combined radiosensitizer administration. Mean tumor concentrations over the 30 min following the end of infusion were 30 and 72 micrograms/g for Ro 03-8799 and SR-2508, respectively. These values would be expected to translate into an approximate single dose sensitizer enhancement ratio of 1.5 to 1.6, offering a significant gain over the enhancement possible with the drugs given alone. The overall advantage will be determined by the maximum dose levels and number of doses possible; the escalation of both parameters is now in progress.

Adolescent↗
PubMed1986

The in vitro effects and cross-resistance patterns of some novel anthracyclines.

A range of new anthracyclines, structurally related to adriamycin (ADM), has been synthesised and studied in vitro. Three compounds described in this paper (Ro 31-1215; Ro 31-1741; Ro 31-2035) are all 4-demethoxyanthracyclines. In the mouse mammary tumour cell line, EMT6/Ca/VJAC, using a 1 h drug exposure followed by colony formation as the response endpoint, we found Ro 31-1215 and Ro 31-1741 to be 2-3 x and 4-7 x more potent then ADM, whilst Ro 31-2035 was 3-4 x less potent. For continuous drug exposure and suppression of population growth as the endpoint, the potency of Ro 31-1741 was similar to that of ADM, whereas that of Ro 31-1215 was 1.5-2 x higher and that of Ro 31-2035 was 10-20 x lower. The potency ratios for continuous drug exposure of a human small cell lung cancer line were similar to those for continuous exposure of EMT6. Variants of the two cell lines selected for resistance to ADM were also studied. These variants also showed considerable resistance to Ro 31-1741 and Ro 31-2035 but much less resistance to Ro 31-1215 (a 9-methyl derivative). A variant of EMT6 made resistant to Ro 31-1215 by continuous growth in this drug was more resistant to ADM than it was to Ro 31-1215. Human cells resistant to ADM contained 6 x less ADM after 24 h exposure than did the parent line, whereas the ratio of drug content for Ro 31-1215 was only 2.

Animals↗
PubMed1986

A clinical phase I toxicity study of Ro 03-8799: plasma, urine, tumour and normal brain pharmacokinetics.

Ro 03-8799 is a lipophilic, basic 2-nitroimidazole of greater potency than misonidazole, which we have administered to 52 patients. The dose-limiting toxicity is an acute central nervous system toxicity with symptoms which include nausea, disorientation, sweating, a feeling of heat and, in one extreme case, coma. Pharmacokinetic analysis was carried out in 31 patients. The mean distribution phase half-life was 44 min and the mean elimination half-life was 6.1 h. Peak concentration was linearly related to dose over the range 0.25 g/m2 to 3 g/m2 with a mean at 1 g/m2 of 15.7 micrograms/ml. Area under the curve was also linearly related to dose and the average whole body clearance was 20.1 l/h. Urinary recovery at 24 h was 31% for the parent compound and 28% for the N-oxide metabolite. The drug is concentrated in normal brain, brain tumour and non-brain tumour to a similar extent, the respective mean tissue/plasma ratios being 381%, 329% and 355%. For a dose of 1 g/m2, tumour concentrations were 1.5 times as high as for misonidazole, and the available in vivo and in vitro sensitisation data predict as improvement of 1.8 and 3.3 times respectively.

Astrocytoma↗
PubMed1986

A comparative study of Ro 03-8799: racemic mixture and enantiomers.

The maximum single dose of the 2-nitroimidazole hypoxic cell radiosensitiser Ro 03-8799 is limited to 1 g/m2 by the occurrence of a well characterised acute syndrome of sweating, nausea and mental changes. In an attempt to increase the tolerable dose, the clinical toxicity of the racemic mixture was compared with that of the R- and S-enantiomers of Ro 03-8799. Twelve patients received escalating alternate doses of racemic mixture and R- or S-enantiomer, the dose levels being 0.25 g/m2, 0.5 g/m2, 0.75 g/m2 and 1.0 g/m2. Careful monitoring of the acute syndrome failed to demonstrate any consistent differences between racemic mixture and either enantiomer. This would suggest that the toxicity is not mediated via any specific central nervous system receptor. It is concluded that separation of Ro 03-8799 into its enantiomers will not enable a clinically useful increase in dosage.

Dose-Response Relationship, Drug↗