Philip Kestell

Synthesis and Biological Evaluation of Novel Analogues of the Pan Class I Phosphatidylinositol 3-Kinase (PI3K) Inhibitor 2-(Difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474)

A structure-activity relationship (SAR) study of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) identified substitution at the 4 and 6 positions of the benzimidazole ring as having significant effects on the potency of substituted derivatives. The 6-amino-4-methoxy analogue displayed a greater than 1000-fold potency enhancement over the corresponding 6-aza-4-methoxy analogue against all three class Ia PI 3-kinase enzymes (p110α, p110β, and p110δ) and also displayed significant potency against two mutant forms of the p110α isoform (H1047R and E545K). This compound was also evaluated in vivo against a U87MG human glioblastoma tumor xenograft model in Rag1(-/-) mice, and at a dose of 50 mg/kg given by ip injection at a qd × 10 dosing schedule it dramatically reduced cancer growth by 81% compared to untreated controls.

Mechanisms of tumor vascular shutdown induced by 5,6-dimethylxanthenone-4-acetic acid (DMXAA): Increased tumor vascular permeability

The novel vascular targeting agent 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) has completed phase 1 clinical trial and has shown tumor antivascular activity in both mice and humans. We have investigated its ability to change tumor vascular permeability, relating it to tumor vascular perfusion and other responses. The murine colon 38 adenocarcinoma was grown in C57Bl wild‐type mice and mice lacking expression of either tumor necrosis factor receptor‐1 (TNFR1−/−) or TNF (TNF−/−). Tumor vascular permeability, as measured by extravasation of albumin‐Evans Blue complexes 4 hr after DMXAA treatment, was significantly increased in tumor tissue in C57Bl, TNFR1−/− and TNF−/− mice but not in normal (skin) tissue. Significant linear relationships were found between increased tumor vascular permeability, decreased functioning tumor blood vessels (measured by Hoechst 33342 staining at 4 hr), increased plasma 5‐hydroxyindole‐3‐acetic acid concentrations (as a measure of serotonin release by platelets) and the degree of induced tumor hemorrhagic necrosis. The results support the hypothesis that DMXAA increases tumor vascular permeability both directly and through the induction of other vasoactive mediators, including TNF. DMXAA might be useful clinically to potentiate the vascular permeability of other anticancer modalities such as cytotoxic drugs, antibodies, drug conjugates and gene therapy.

Thalidomide pharmacokinetics and metabolite formation in mice, rabbits, and multiple myeloma patients.

Purpose: Thalidomide has a variety of biological effects that vary considerably according to the species tested. We sought to establish whether differences in pharmacokinetics could form a basis for the species-specific effects of thalidomide. Experimental Design: Mice and rabbits were administered thalidomide (2 mg/kg) p.o. or i.v., and plasma concentrations of thalidomide were measured after drug administration using high performance liquid chromotography. Plasma samples from five multiple myeloma patients over 24 hours after their first dose of thalidomide (200 mg) were similarly analyzed and all data were fitted to a one-compartment model. Metabolites of thalidomide in plasma were identified simultaneously using liquid chromatography-mass spectrometry. Results: Plasma concentration-time profiles for the individual patients were very similar to each other, but widely different pharmacokinetic properties were found between patients compared with those in mice or rabbits. Area under the concentration curve values for mice, rabbits, and multiple myeloma patients were 4, 8, and 81 μmol/L · hour, respectively, and corresponding elimination half-lives were 0.5, 2.2, and 7.3 hours, respectively. Large differences were also observed between the metabolite profiles from the three species. Hydrolysis products were detected for all species, and the proportion of hydroxylated metabolites was higher in mice than in rabbits and undetectable in patients. Conclusions: Our results show major interspecies differences in the pharmacokinetics of thalidomide that are related to the altered degree of metabolism. We suggest that the interspecies differences in biological effects of thalidomide may be attributable, at least in part, to the differences in its metabolism and hence pharmacokinetics.

Plasma disposition, metabolism and excretion of the experimental antitumour agent 5,6-dimethylxanthenone-4-acetic acid in the mouse, rat and rabbit

Abstract 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), an experimental antitumour agent currently undergoing phase I clinical trial, has a maximum tolerated dose (MTD) in male BDF1 mice of 99 μmol/kg. We have found the male Sprague-Dawley rat and the New Zealand White rabbit to have greater tolerance to DMXAA, with MTDs being 990 and 330 μmol/kg, respectively. To investigate the causes of this difference, we measured plasma and urine DMXAA concentrations by high-performance liquid chromatography (HPLC) after single i.v. bolus injections of 99 and 990 μmol/kg in the rat and following a bolus dose of 99 μmol/kg and a 10-min infusion of 330 μmol/kg in the rabbit. Following administration of DMXAA at the MTD in the mouse, rat and rabbit the maximal concentrations were 600, 2,200 and 1,708 μM, respectively, whereas areas under the concentration-time curves were 2,400, 19,000 and 2,400 μMh, respectively, for unchanged DMXAA. Data obtained for mice and rabbits were satisfactorily fitted to a two-compartment model with Michaelis-Menten kinetics. DMXAA was highly bound to plasma proteins, with the highest degree of binding being found in the rabbit. A small proportion of the total dose (7.8%, 0.6% and 12.4%, respectively) was excreted unchanged in urine over 24 h. This proportion increased (to 11.6%, 3.5% and 72.4%, respectively) following alkaline hydrolysis, suggesting the presence of glucuronide metabolites. Examination of rat and mouse urine by HPLC revealed the presence of two metabolites, which were characterized by mass spectrometry and nuclear magnetic resonance to be the acyl glucuronide of DMXAA and 6-(hydroxymethyl)-5-methylxanthenone-4-acetic acid. Thus, both mice and rats metabolise DMXAA by similar pathways. The results demonstrate considerable interspecies variations in tolerance to DMXAA that cannot be explained by differences in pharmacokinetics.

5,6-Dimethylxanthenone-4-Acetic Acid (DMXAA): a New Biological Response Modifier for Cancer Therapy

The investigational anti-cancer drug5,6-dimethylxanthenone-4-acetic acid(DMXAA) was developed by the AucklandCancer Society Research Centre (ACSRC). Ithas recently completed Phase I trials inNew Zealand and UK under the direction ofthe Cancer Research Campaigns Phase I/IIClinical Trials Committee. As a biologicalresponse modifier, pharmacological andtoxicological properties of DMXAA areremarkably different from most conventionalchemotherapeutic agents. Induction ofcytokines (particularly tumour necrosisfactor (TNF-α), serotonin and nitricoxide (NO)), anti-vascular andanti-angiogenic effects are considered tobe major mechanisms of action based on invitro and animal studies. In cancerpatients of Phase I study, DMXAA alsoexhibited various biological effects,including induction of TNF-α,serotonin and NO, which are consistent withthose effects observed in in vitroand animal studies. Preclinical studiesindicated that DMXAA had more potentanti-tumour activity compared toflavone-8-acetic acid (FAA). In contrast toFAA that did not show anti-tumour activityin cancer patients, DMXAA (22 mg/kg byintravenous infusion over 20 min) resultedin partial response in one patient withmetastatic cervical squamous carcinoma in aPhase I study where 65 cancer patients wereenrolled in New Zealand. The maximumtolerated dose (MTD) in mouse, rabbit, ratand human was 30, 99, 330, and 99 mg/kgrespectively. The dose-limiting toxicity ofDMXAA in cancer patients included acutereversible tremor, cognitive impairment,visual disturbance, dyspnoea and anxiety.The plasma protein binding and distributioninto blood cells of DMXAA are dependent onspecies and drug concentration. DMXAA isextensively metabolised, mainly byglucuronidation of its acetic acid sidechain and 6-methylhydroxylation, givingrise to DMXAA acyl glucuronide (DMXAA-G),and 6-hydroxymethyl-5-methylxanthenone-4-aceticacid (6-OH-MXAA), which are excreted intobile and urine. DMXAA-G has been shown tobe chemically reactive, undergoinghydrolysis, intramolecular migration andcovalent binding. Studies have indicatedthat DMXAA glucuronidation is catalysed byuridine diphosphateglucuronosyltransferases (UGT1A9 andUGT2B7), and 6-methylhydroxylation bycytochrome P450 (CYP1A2). Non-linear plasmapharmacokinetics of DMXAA has been observedin animals and patients, presumably due tosaturation of the elimination process andplasma protein binding. Species differencesin DMXAA plasma pharmacokinetics have beenobserved, with the rabbit having thegreatest plasma clearance, followed by thehuman, rat and mouse. In vivo disposition studies inthese species didnot provide an explanation for thedifferences in MTD. Co-administration ofDMXAA with other drugs has been shown toresult in enhanced anti-tumour activity andalterations in pharmacokinetics, asreported for the combination of DMXAA withmelphalan, thalidomide, cyproheptadine, andthe bioreductive agent tirapazamine, inmouse models. Species-dependentDMXAA-thalidomide pharmacokineticinteractions have been observed.Co-administration of thalidomidesignificantly increased the plasma area ofthe plasma concentration-time curve (AUC)of DMXAA in mice, but had no effect onDMXAAs pharmacokinetics in the rat. Itappears that the pharmacological andtoxicological properties of DMXAA as a newbiological response modifier are unlikelyto be predicted based on preclinicalstudies. Similar to many biologicalresponse modifiers, DMXAA alone did notshow striking anti-tumour activity inpatients. However, preclinical studies ofDMXAA-drug combinations indicate that DMXAAmay have a potential role in cancertreatment when co-administered with otherdrugs. Further studies are required toexplore the molecular targets of DMXAA andmechanisms for the interactions with otherdrugs co-administered during combinationtreatment, which may allow for theoptimisation of DMXAA-based chemotherapy.

Measurement of plasma 5-hydroxyindoleacetic acid as a possible clinical surrogate marker for the action of antivascular agents

BACKGROUND Serotonin (5HT), a naturally occurring vasoactive substance, is released from platelets into plasma under various pathological conditions. Recently, anticancer drugs that act by selectively disrupting tumour blood flow have been found to increase plasma 5HT concentrations in mice. Two such antivascular agents, flavone acetic acid (FAA) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), have completed Phase I clinical trial and raise the important question of whether suitable surrogate markers for antivascular effects can be identified. METHODS 5HT is unstable to storage, precluding routine clinical assay, but the 5HT metabolite, 5-hydroxyindoleacetic acid (5HIAA) accumulates in plasma following 5HT release and is a more suitable marker because of its greater stability. We have developed an automated procedure for the assay of the low concentrations of 5HIAA found in humans by combining solid-phase extraction with high-performance liquid chromatography (HPLC). RESULTS Efficient separation of 5HIAA from possible interfering substances in human plasma, including a variety of pharmaceutical agents, was achieved on C18 columns using cetyltrimethylammonium bromide (CETAB) as an organic modifier. Adequate precision, accuracy and sensitivity were achieved by electrochemical detection (ECD) at +400 mV. Analysis of plasma from two patients treated with DMXAA in a Phase I trial demonstrated DMXAA-induced elevation of plasma 5HIAA with a time course similar to that previously described in mice. CONCLUSIONS Measurement of changes in plasma 5HIAA provides a new approach to the monitoring of therapies with an antivascular effect. The assay is sensitive to dietary sources of 5HT, which should be minimised.

The adsorption of a range of dietary carcinogens by α-cellulose, a model insoluble dietary fiber

Abstract One of the ways dietary fibers may protect against colorectal cancer is by adsorbing carcinogens and carrying them out of the digestive tract, thus lessening interaction of the carcinogens with the colonic tissue. We investigated this mechanism of action by testing in vitro the abilities of a range of carcinogens, including known animal colon carcinogens, to adsorb to α-cellulose, which we have used as a model insoluble dietary fiber. The carcinogens were N-nitroso-N-methylurea (NMU), benzo[a]pyrene (B[a]P) and a number of heterocyclic aromatic amines which have been found in heated foods. It was found that the ability of a carcinogen to adsorb to α-cellulose is strongly related to the hydrophobicity of the carcinogen measured as the calculated logarithm of the partition coefficient between 1-octanol and water (C log P). The hydrophilic carcinogen, NMU, (C log P = −0.204), adsorbed only poorly, whereas the very hydrophobic carcinogen, B[a]P, (C log P = 6.124), adsorbed strongly. Carcinogens with intermediate hydrophobicities showed intermediate abilities to adsorb.

Plasma pharmacokinetics of the antitumour agents 5,6-dimethylxanthenone-4-acetic acid, xanthenone-4-acetic acid and flavone-8-acetic acid in mice

SummaryAlthough the antitumour agent flavone-8-acetic acid (FAA) exhibits remarkable activity against murine solid tumours, its clinical use has a number of pharmacological drawbacks, including low dose potency and dose-dependent pharmacokinetics. Xanthenone-4-acetic acid (XAA) and its 5,6-dimethyl derivative (5,6-MeXAA) were synthesised during a search for better analogues of FAA. The maximal tolerated doses (MTDs) of 5,6-MeXAA, XAA and FAA in BDF1 mice were 99, 1,090 and 1,300 μmol/kg, respectively. At the MTD, 5,6-MeXAA displayed the following pharmacokinetic properties: maximal plasma concentration, 600 μM; mean residence time, 4.9 h; AUC, 2,400 μmol h l−1; and volume of steady-state distribution, 0.2 l/kg. All compounds displayed nonlinear elimination kinetics at the MTD, but when the logarithm of the AUC was plotted against that of the delivered dose, the slope of the regression line for 5,6-MeXAA was found to be 1.2 as opposed to 1.4 for XAA and 1.98 for FAA. 5,6-MeXAA thus showed only a slight deviation from dose-independent kinetics. 5,6-MeXAA bound to plasma proteins in a manner similar to that exhibited by FAA, although the plasma concentration of free drug was lower for the former than for the latter. As a consequence, the calculated maximal free drug concentration for 5,6-MeXAA in plasma was 23 times lower than that for FAA.

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.

Modulation of the pharmacokinetics of the antitumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in mice by thalidomide.

Background: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), an investigative drug currently in clinical trial, acts on tumour vasculature through the induction of cytokines. Coadministration of thalidomide, a modulator of cytokine production, potentiates the antitumour activity of DMXAA against the murine Colon 38 carcinoma in mice. We wished to determine whether alteration of the pharmacokinetics of DMXAA by thalidomide could provide an explanation for this potentiation. Results: Coadministration of thalidomide to Colon 38 tumour-bearing mice significantly (P < 0.05) increased the elimination half-life (t1/2) of DMXAA in plasma (413 μmol/l), liver (132 μmol/l), and spleen (77 μmol/l), and significantly (P < 0.05) increased DMXAA concentrations in Colon 38 tumour tissue (0.25–4.5 h). l-Thalidomide had a greater effect on DMXAA elimination (P < 0.01) than did d-thalidomide or the racemate. Coadministration of thalidomide increased the area under the concentration-time curve (AUC) of DMXAA by 1.8-fold in plasma, liver and spleen, and by 3.0-fold in tumour. Bile from mice given thalidomide and DMXAA contained substantially lower amounts of the glucuronide metabolite of DMXAA (DMXAA-G) than did bile from mice given DMXAA alone. Conclusion: Glucuronidation is a major excretory pathway for DMXAA in the mouse. Thalidomide, probably as the l-form, decreases the rate of elimination of DMXAA from plasma, spleen, liver and tumour by altering the rate of glucuronidation. The reduction in the elimination of DMXAA by thalidomide may lead to a selective increase in exposure of tumour tissue to drug, providing a basis for its potentiation of antitumour activity.