Iain G. C. Robertson

Metabolism of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide in cancer patients undergoing a phase I clinical trial.

N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (DACA) is an experimental antitumour agent that has just completed phase I clinical trials in New Zealand and the United Kingdom. Urine (0–72 h) was analysed from 20 patients receiving DACA infused over 3 h (dose range 60–1000 mg/m2, the latter being the highest dose achieved in the trial). Aliquots were analysed for DACA and its metabolites by high-performance liquid chromatography (HPLC). Over 72 h, 44 ± 5% (range 20–60%) of the dose was recovered in the urine, with 0.8 ± 0.3% (range 0–3.1%) occurring as DACA. The major urinary metabolite was DACA-N-oxide-9(10H)acridone, accounting for 34 ± 3% of the dose. Minor metabolites were identified as N-mono- methyl-DACA-9(10H)acridone (2.0 ± 0.5%), DACA-9(10H)acridone (3.3 ± 0.5%), N-monomethyl-DACA (0.2 ± 0.1%) and DACA-N-oxide (0.5 ± 0.1%). No ring-hydroxylated metabolite was detected. The urinary excretion of metabolites was greatest over 0–6 h in most patients. The composition of urinary metabolites was also independent of the delivered dose. Plasma was sampled at intervals throughout the infusion and at time points up to 48 h post-administration. The major plasma metabolites observed were DACA-9(10H)acridone and DACA-N-oxide-9(10H)acridone. These results indicate that, based on urinary excreted metabolites, the major biotransformation reactions for DACA in humans involve N-oxidation of the tertiary amine side chain and acridone formation, both of which appear to be detoxication reactions.

Pharmacokinetics and toxicity of the antitumour agentN-[2-(dimethylamino)ethyl]acridine-4-carboxamide after i.v. administration in the mouse

SummaryThe pharmacokinetics, tissue distribution and toxicity of the antitumour agentN-[2-(dimethylamino)ethyl]acridine-4-carboxamide(AC) were studied after i.v. administration to mice. Over the dose range of 9–121 μmol/kg (3–40 mg/kg), AC displayed linear kinetics with the following model-independent parameters: clearance (C), 21.0±1.9 l h−1 kg−1; steady-state volume of distribution (Vss), 11.8±1.4 l/kg; and mean residence time (MRT), 0.56±0.02 h. The plasma concentration-time profiles for AC fitted a two-compartment model with the following parameters:Cc, 19.4±2.3 l h−1 kg−1; Vc, 7.08±1.06 l/kg;t1/2α 13.1±3.5 min; andt1/2Z, 1.60±0.65 h. AC displayed moderately high binding in healthy mouse plasma, giving a free fraction of 15.9%–25.3% over the drug concentration range of 1–561 μM. After the i.v. administration of 30 μmol/kg [3H]-AC, high radioactivity concentrations were observed in all tissues (especially the brain and kidney), showing a hight1/2c value (37–59 h). At 2 min (first blood collection), the AC concentration as measured by high-performance liquid chromatography (HPLC) comprised 61% of the plasma radioactivity concentration (expressed as AC equivalents/l). By 48 h, 73% of the dose had been eliminated, with 26% and 47% of the delivered drug being excreted by the urinary and faecal routes, respectively; <1% of the total dose was excreted as unchanged AC in the urine. At least five distinct radiochemical peaks were distinguishable by HPLC analysis of plasma extracts, with some similar peaks appearing in urine. The 121-μmol/kg dose was well tolerated by mice, with sedation being the only obvious side effect and no significant alterations in blood biochemistry or haematological parameters being recorded. After receiving a dose of 152 μmol/kg, all mice experienced clonic seizures for 2 min (with one death occuring) followed by a period of sedation that lasted for up to 2h. No leucopenia occurred, but some mild anaemia was noted. There was no significant change in blood biochemistry. A further 20% increase in the i.v. dose (to 182 μmol/kg) resulted in mortality, with death occurring within 2 min of AC administration.

Intraperitoneal administration of the antitumour agentN-[2-(dimethylamino)ethyl]acridine-4-carboxamide in the mouse: bioavailability, pharmacokinetics and toxicity after a single dose

SummaryThe pharmacokinetics, tissue distribution and toxicity of the antitumour agentN-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (AC) were studied after i.p. administration of [3H]-AC (410 μmol/kg) to mice. The latter is the optimal single dose for the cure of advanced Lewis lung tumours. AC was rapidly absorbed into the systemic circulation after i.p. administration, with the maximal concentration (Cmax) occurring at the first time point (5 min). There was no reduction in bioavailability as compared with previous i.v. studies, but the shape of the plasma concentration-time profile was considerably different, reflecting a 3-fold lowerCmax value (20.9±3.6 μmol/l) and a longert1/2 value (2.7±0.3 h) as compared with that observed after i.v. administration (1.6±0.6 h). Model independent pharmacokinetic parameters after i.p. administration were: clearance (C), 17.5 l h−1 kg−1; steady-state volume of distribution (Vss), 14.1 l/kg; and mean residence time (MRT), 1.46 h. High but variable tissue uptake of AC was observed, with tissue/plasma AUC ratios being 5.7 for heart, 8.4 for brain, 18.9 for kidney and 21.0 for liver but with similar eliminationt1/2 values ranging from 1.3 to 2.7 h. All radioactivity profiles in plasma and tissues were greater than the respective parent AC profiles and showed prolonged eliminationt1/2 values ranging from 21 h in liver to 93 h in brain. However, tissue/plasma radioactivity AUC ratios were near unity, ranging from 0.7 to 1.57, with the exception of the gallbladder (15.6), which contained greater amounts of radioactivity. By 48 h, approximately 70% of the total dose had been eliminated, with the faecal to urinary ratio being approximately 2:1. This i.p. dose was well tolerated by mice, with sedation being the only obvious side effect. No major change was observed in blood biochemistry or haematological parameters. Comparisons ofCmax,tmax and AUC values determined for AC in brain after its i.p. and i.v. administration suggest that the reduction in acute toxicity after i.p. administration is not due to reduced exposure of the brain to AC as measured by AUC but may be associated with the lowerCmax value or the slower rate of entry of AC into the brain after i.p. administration.

Plasma Protein Binding of the Experimental Antitumour Agent Acridine‐4‐carboxamide in Man, Dog, Rat and Rabbit

Abstract— The plasma binding of N‐[2‐(dimethylamino)ethyl]acridine‐4‐carboxamide (AC) was investigated in‐vitro by equilibrium dialysis for 3 h at 37°C against isotonic phosphate buffer (pH 7·35) using [3H]AC. There were significant species differences with the smallest % free fraction (mean ± s.d.) occurring in human plasma (3·4 ± 0·2), followed by dog (8·1 ±0·4), mouse (14·8 ± 0·8), rat (16·3 ± 0·9) and rabbit (20·2 ± 0·7). In plasma from healthy individuals (n = 5), the % free fraction ranged from 2·7 to 3·8. In physiological solutions of human proteins, the greatest binding was observed for α‐acid glycoprotein (AAG) (0·75 g L−1) with a mean free fraction of 24·1 ± 2·2%, followed by albumin (40 g L−1) with 31·6 ± 0·7 and 39·8 ± 2·5% for fatty‐acid‐free and globulin‐free, respectively. There was also some binding to globulins (5 g L−1) with a mean % free fraction of 70·3 ± 1·6 and 84·8 ± 2·2 for Conns fraction I and IV, respectively. Binding data from the displacement of [3H]AC by increasing concentrations of AC in human AAG (0·75 g L−1) or albumin solution (40 g L−1) indicated that AAG had 10‐fold greater binding affinity for AC (Ka, 7·8 × 104 m−1) compared with albumin (Ka, 6·8 × 103 m−1). In human plasma enriched with AAG there was a significant negative linear correlation (r = 0·932; P < 0·001) between % AC free fraction and increasing AAG concentration over the range 0·6–4·5 g L−1. Small but significant (P < 0·05) increases in AC free fraction occurred in the presence of various metabolites (50 and 100 μm) but, of those tested, only N‐monomethyl‐acridine carboxamide increased the free fraction to the same extent as parent AC.

A comparison of the requirements for antitumour activity and antibacteriophage lambda activity for a series of non-intercalative DNA-binding agents☆

A series of non-intercalative DNA-binding agents, comprising mainly bisquaternary ammonium heterocyclic compounds, has been found to inhibit strongly the production of bacteriophage lambda following its induction in Escherichia coli. The inhibition is much greater than that found with a number of DNA intercalating agents, including 9-aminoacridine, ethidium and Daunorubicin. The inhibition correlated significantly with antitumour effect, as measured in a life extension assay with L1210 leukaemia. Activity in both biological systems demanded the presence of strongly charged groups and a rigid co-planar aromatic skeleton, these requirements being almost identical to those needed to displace ethidium efficiently from DNA in a simple assay system. It is suggested that biological activity is associated with the ability of these agents to bind in the minor groove of the DNA double helix. Data on the antibacteriophage action of one of these agents suggests possible models for antitumour activity.

Evaluation of potential health effects of 10 kHz magnetic fields: A short-term mouse toxicology study

A high-frequency inductive power distribution (HID) technology has been developed that generates sinusoidal magnetic fields at a frequency of 10 kHz. In typical industrial applications, field intensities in the order of 0.2 mT can be expected between the current-carrying coils. Because the possible health effects of 10 kHz sinusoidal magnetic fields of this type had never been investigated, a broad evaluation of possible effects on animal health was made in a preliminary 14 day acute study and in a 90 day subchronic study using male and female B6C3F1 mice. Exposures were at 0.08, 0.28, and 1.0 mT vs. a background exposure of 3.7 microT and were essentially continuous. These studies failed to demonstrate any health effects that can be clearly related to the magnetic field exposure. No changes in animal behaviour or indications of morbidity were detected during the initial exposure to the fields. There were no significant differences in body weight between exposed and unexposed (control) mice at any time in the study, and the clinical chemistry and hematology parameters were essentially unchanged. Although minor differences in some clinical chemistry and hematology parameters were seen between control and exposure groups, the lack of exposure dependence, the lack of consistency between sexes, and the lack of correspondence with the results of the two studies all suggest that these were chance associations. Even if the changes were real, the magnitude of the changes was very small and does not indicate serious biological effects. Finally, all organs were macroscopically and microscopically normal except for isolated, generally mild, histological lesions and lesions that were ascribed to fighting among males. There was no obvious association with field intensity.

Tumour profile ofN-[2-(dimethylamino)ethyl]acridine-4-carboxamide after intraperitoneal administration in the mouse

N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (AC) is an experimental antitumour agent that is being considered for phase I trials. After i.p. administration of 150 mg/kg [3H]-AC to tumour-bearing mice, AC was absorbed rapidly into the plasma and tissues such as the heart, liver, kidney and brain but more slowly into the s.c. tumour. The maximal AC concentration (86±36 μmol/kg) in the tumour occurred at 35–60 min and was 3-fold the maximal plasma concentration, which occurred at 15 min. Although higher maximal concentrations were observed in other tissues, these concentrations fell rapidly in parallel with plasma concentrations. In contrast, AC concentrations in the tumour remained elevated, thet1/2 value (16.3 h) and mean residence time (MRT, 9.5 h) being prolonged in comparison with those in the plasma and other tissues (t1/2 range, 1.0–2.9 h; MRT, 1.2–1.4 h). AC concentrations were not detectable by our high-performance liquid chromatographic (HPLC) method (limit of detection, 0.02 μmol/l) in the plasma or other tissues at 24 or 48 h after administration but were measurable in the tumour (1.6±0.8 and 0.6±0.3 μmol/kg, respectively). Radioactivity concentrations in the plasma, tissues and tumour were very variable but were greater than the corresponding levels of unchanged parent AC. By 24 h, radioactivity concentrations in the plasma, tissues and tumour had fallen to similar levels with prolonged elimination profiles. Thus, the exposure of the s.c. implanted tumour to a threshold AC concentration for a prolonged time (>24 h) may partly explain the greater efficacy of AC against this tumour, whereas the shorter period of exposure of blood and other tissues may explain its low haematological toxicity.

The relationship between lipophilic-hydrophilic balance, uptake and anti-bacteriophage lambda activity of experimental anti-tumour bisquaternary salts

The uptake by Escherichia coli of a series of bisquaternary experimental anti-tumour agents (quinolinium 4-[p-9(-pyridylamino)phenylcarbamoyl]-aniline-bisalkyl dibromides) has been measured both by association of radiolabelled compounds and their inhibition of the vegetative replication of bacteriophage lambda (after heat inactivation of the phage repressor) as a measure of biologically effective intracellular drug concentration. Uptake of these compounds was correlated with biological effect, and was a function of both incubation temperature and the lipophilic-hydrophilic balance of the compound. At 30 degrees C uptake was drug concentration-dependent and was not readily reversible. No saturation of uptake was apparent over the concentration range tested. Preliminary experiments indicated that time-dependent drug uptake was also related to growth inhibition in cultured L1210 murine leukaemia cells. These results are consistent with the hypothesis that uptake occurs by diffusion across the plasma membrane followed by strong binding to cell constituents such as DNA. The approximate range of uptake of the most active compounds, using an external drug concentration of 1 microM, are 100 and 2400 molecules/s respectively for bacteria and murine leukemia cells. For bacteria, the uptake of approx. 2 X 10(5) molecules of drug/cell inhibits the yield of phage lambda by 90%.

Enzymatic Detoxication of Tumorigenic Bay-Region Diol-Epoxides of Polycyclic Aromatic Hydrocarbons by Conjugation with Glutathione

Polycyclic aromatic hydrocarbons (PAH), such as benzo(a)pyrene (BP), benz(a)anthracene (BA) and chrysene (C), are widely distributed contaminants in the environment and proven tumorigens in experimental animals. Epidemiological data indicate a role for PAH also in the etiology of certain human tumors1. PAH require metabolic transformation to electrophilic intermediates to exert their toxic effects, most probably through covalent binding of these intermediates to critical targets in DNA. BP is activated through the action of cytochrome P-450-linked monooxygenases and epoxide hydrolase to diastereomeric trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-BP (anti- and syn-BPDE). The (+)-enantiomer of anti-BPDE, with R,S,S,R-absolute configuration, is the most tumorigenic one of the isomers in animals2. Similar results have been obtained with the equivalent diol-epoxides of BA and C3. Furthermore, covalent binding of (+)-anti-BPDE to the exocyclic nitrogen of deoxyguanosine in DNA of target tissues is closely correlated with tumor formation4. Thus, it is likely that enzymatic and non-enzymatic processes that prevent intracellular accumulation of PAH diol-epoxides will counteract DNA-damage and resulting consequences.