Neville Willmott

Pharmacokinetics and pharmacodynamics of locoregional 5 fluorouracil (5FU) in advanced colorectal liver metastases

By measuring peripheral drug levels in plasma, the effect of combining albumin microspheres with angiotensin II on systemic exposure to 5FU when administered by bolus injection into the hepatic artery in patients with advanced colorectal liver metastases has been assessed. The results suggest that despite hepatic arterial administration of 5FU, there was no reduction in systemic exposure when compared with that associated with intravenous injection of the same dose. Neither albumin microspheres nor angiotensin II appeared to improve the regional advantage. There have been a number of reports relating the plasma levels of cytotoxic agents with pharmacodynamic parameters. We have shown significant direct correlations between 5FU clearance and 1 week post treatment platelet and white cell counts, and an inverse relationship between the area under the 5FU plasma concentration-time curve (AUC) and 1 week post treatment platelet count and white cell count respectively.

Disposition kinetics of adriamycin, adriamycinol and their 7-deoxyaglycones in AKR mice bearing a sub-cutaneously growing Ridgway Osteogenic Sarcoma (ROS)

Disposition kinetics of Adriamycin (ADR), adriamycinol (AOL) and their 7-deoxyaglycones (ADR-DONE and AOL-DONE) have been studied in AKR mice bearing a s.c. growing ROS tumour after i.v. administration of 10 mg/kg. ADR and its metabolites were extracted from tissues by two different methods, separated and identified by HPLC. Tissue 7-deoxyaglycones were isolated, purified and then identified by HPLC, TLC and mass spectrometry. Kinetic profiles of ADR showed rapid equilibration of the drug with well perfused tissues but a slower and complex equilibration of the drug with the ROS tumour. Serum and tissue profiles of AOL were similar to the parent drug. From the kinetic profiles of the 7-deoxyaglycones it appeared that in the tissues their formation was rapid, with ADR-DONE always appearing first. Maximum concentrations of ADR-DONE were reached in the liver and heart only 10 min after drug administration. Estimated half lives of ADR-DONE were in liver, 1.1 hr and in heart, 2.8 hr and for AOL-DONE in liver, 5.4 hr, in heart, 5.1 hr and in serum, 4.1 hr.

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, Students 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.

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 Students 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.

Increased anti-tumor effect of adriamycin-loaded albumin microspheres is associated with anaerobic bioreduction of drug in tumor tissue

Anti-tumor activity and fate of adriamycin incorporated into biodegradable albumin microspheres was examined in vivo after direct intratumoral injection. Adriamycin in microspherical form displayed superior anti-tumor activity to a comparable dose of drug in solution. This was associated at later time points (40 hr, 50 hr and 72 hr after injection) with higher median parent drug concentrations in tumor tissue (4.1, 3.6, 2.6 micrograms/g respectively for microspheres and 1.6, 1.7 and 1.0 micrograms/g for solution) and the consistent detection of 7-deoxyaglycone metabolites, end products of reduction of adriamycin under anaerobic conditions (1.1, 1.0, 1.0 micrograms/g respectively for microspheres and less than 0.1 micrograms/g at all time points for solution). It is generally considered that the redox properties of anthracyclines are responsible for their toxicity to normal tissues whereas other mechanisms are responsible for antineoplastic activity. In this study we show that inducing metabolism of Adriamycin via reductive pathways is associated with increased anti-tumor effect.

Regional delivery of microspheres to liver metastases: the effects of particle size and concentration on intrahepatic distribution

There is increasing interest in the use of microspheres, loaded with chemotherapeutic agents, for regional therapy to hepatic metastases. It is necessary to deliver these particles predominately to tumour rather than to normal liver. This study investigates factors influencing the distribution of regionally injected microspheres. Discreet tumour was induced in rats by subcapsular hepatic inoculations of HSN cells. At 20 days, 12.5 microns, 25 microns or 40 microns diameter, radiolabelled albumin microspheres were administered, in various concentrations, via the gastroduodenal artery. Tumour to normal liver microsphere distribution ratios were determined and median values ranged from 0.1 (0.2 mg ml-1 12.5 microns microspheres) to 1.8 (20 mg ml 40 microns microspheres). Concentrated suspensions (20 mg ml-1) of large microspheres (40 microns) produced the most favourable tumour to normal liver distribution ratios. These results not only have implications for the therapeutic administration of microspheres but also for their use in blood-flow studies.

Intra-arterial administration of adriamycin-loaded albumin microspheres for locally advanced breast cancer.

Regional chemotherapy is an attractive but underevaluated method of treating locally advanced breast cancer. We have combined two novel methods of targeting by delivering a single pulse of adriamycin-loaded albumin microspheres down a radiologically placed internal mammary artery catheter. A complete response was observed and prolonged local control achieved.

Target organ disposition and plasma pharmacokinetics of doxorubicin incorporated into albumin microspheres after intrarenal arterial administration.

We synthesized doxorubicin (Adriamycin, Adria Laboratories, Columbus, OH)‐loaded human albumin microspheres (containing approximately 1% doxorubicin w/w) between 15 and 20 μm in diameter. Intrarenal arterial administration of99mTC‐labeled microspheres demonstrated a high renal entrapment ratio (97% of recovered radioactivity). The pharmacokinetics and metabolism of doxorubicin are different when it is administered in microspherical form. Peak plasma levels are lower (16 ng/ml versus, 135 ng/ml) compared with treatment by a doxorubicin solution. Histologic studies showed that the micro‐spheres were trapped within capillaries and small arterioles in the renal vascular arcade. It is apparent that chemoembolization with doxorubicin‐loaded microspheres significantly reduces systemic exposure to the antineoplastic agent, and maintains intrarenal drug levels.

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.

The pharmacokinetics of 5-fluorouracil administered by arterial infusion in advanced colorectal hepatic metastases.

The pharmacokinetics of 5-fluorouracil (5FU) following its administration via the hepatic artery in conjunction with biodegradable albumin microspheres and angiotensin II have been studied. Peripheral venous concentrations of 5FU are lower and plasma clearance values higher following intrahepatic arterial administration compared with a similar dose administered by intravenous infusion over both 2 h and 24 h. For the 2 h drug infusions, plasma 5FU concentrations following co-treatment with angiotensin II and microspheres via the hepatic artery were intermediate between those of arterial and venous infusions of 5FU alone. There was a trend towards the peak plasma drug concentrations and the area under the plasma concentration-time curve (AUC) being significantly lower following co-treatment with angiotensin II and microspheres compared with intra-arterial and intravenous infusions of 5FU over 24 h. Co-administration of 5FU, angiotensin II and microspheres via the hepatic artery may reduce drug exposure in the systemic compartment and therefore may increase the therapeutic ratio of 5FU administration via the hepatic artery.