Cellular glutathione as a determinant of the sensitivity of colorectal tumour cell-lines to ZD2767 antibody-directed enzyme prodrug therapy (ADEPT).

ZD2767P, a nitrogen mustard glutamate prodrug, is currently being evaluated in Phase 1 clinical trials of antibody directed enzyme prodrug therapy (ADEPT). There was no significant relationship between basal glutathione (GSH) concentration and sensitivity to ZD2767P + carboxpeptidase G2 (CPG2) in colorectal tumour cell-lines. Depletion of intracellular GSH using buthionine sulfoximine (BSO) resulted in only a modest potentiation of ZD2767P + CPG2 activity and hence BSO is unlikely to markedly enhance the activity of this ADEPT treatment.

Impact of polyglutamation on sensitivity to raltitrexed and methotrexate in relation to drug-induced inhibition of de novo thymidylate and purine biosynthesis in CCRF-CEM cell lines.

The aim of this study was to investigate the influence of folylpolyglutamyl synthetase (FPGS) activity on the cellular pharmacology of the classical antifolates raltitrexed and methotrexate (MTX) using two human leukemia cell lines, CCRF-CEM and CCRF-CEM:RC2Tomudex. Cell growth inhibition and drug-induced inhibition of de novo thymidylate and purine biosynthesis were used as measures of the cellular effects of the drugs. CCRF-CEM:RC2Tomudex cells had <11% of the FPGS activity of CCRF-CEM cells, whereas MTX uptake and TS activity were equivalent. In CCRF-CEM:RC2Tomudex cells, MTX polyglutamate formation was undetectable after exposure to 1 microM [3H]MTX for 24 h. After exposure to 0.1 microM raltitrexed, levels of total intracellular raltitrexed-derived material in CCRF-CEM:RC2Tomudex cells were 30- to 50-fold lower than in the CCRF-CEM cell line. CCRF-CEM: RC2Tomudex cells were >1000-fold resistant to raltitrexed and 6-fold resistant to lometrexol but sensitive to MTX and nolatrexed when exposed to these antifolates for 96 h. After 6 h of exposure, CCRF-CEM cells retained sensitivity to MTX and raltitrexed but were less sensitive to lometrexol-mediated growth inhibition. In contrast, CCRF-CEM: RC2Tomudex cells were markedly insensitive to raltitrexed, lometrexol, and to a lesser degree, MTX. Simultaneous measurement of de novo thymidylate and purine biosynthesis revealed 90% inhibition of TS activity by 100 nM MTX in both cell lines, whereas inhibition of de novo purine synthesis was only observed in CCRF-CEM cells, and only after exposure to 1000 nM MTX. Ten nM raltitrexed induced >90% inhibition of TS activity in CCRF-CEM cells, whereas in CCRF-CEM:RC2Tomudex cells, there was no evidence of inhibition after exposure to 1000 nM raltitrexed. These studies demonstrate that polyglutamation is a critical determinant of the cellular pharmacology of both raltitrexed and MTX, markedly influencing potency in the case of raltitrexed and locus of action in the case of MTX.

Phase I studies with the nonclassical antifolate nolatrexed dihydrochloride (AG337, THYMITAQ) administered orally for 5 days.

Phase I studies of p.o. administered nolatrexed dihydrochloride (AG337, THYMITAQ), a nonclassical thymidylate synthase inhibitor, were performed to establish the maximum tolerated dose and a recommended dose for Phase II studies. The bioavailability and pharmacokinetic and pharmacodynamic properties of oral nolatrexed were also studied. Forty-five patients were treated with oral nolatrexed every 6 h for 5 days at doses of 288-1000 mg/m2/day. The bioavailability of the oral preparation was determined, and the effect of a standard meal on nolatrexed absorption was investigated at a dose of 800 mg/m2/day. Nolatrexed plasma concentrations were analyzed by high-performance liquid chromatography. Nolatrexed was rapidly absorbed with a median bioavailability of 89% (range 33-116%), with 88% of patients above 70%. The dose-limiting toxicities were gastrointestinal, and the recommended Phase II oral dose was 800 mg/m2/day. After a standard meal, the peak plasma nolatrexed concentration achieved was lower (median, 8.3 microg/ml versus 15.0 microg/ml; P = 0.001), and the time taken to reach the peak was longer (median, 180 min versus 45 min; P = 0.00003), but the trough concentration was higher (median, 3.6 microg/ml versus 2.1 microg/ml; P = 0.004) when compared with the fasted state. The area under the nolatrexed plasma concentration versus time curve was not affected by food. Average trough nolatrexed concentration, but not dose, was significantly related to the % decrease in both thrombocytes (r2 = 0.58; C50 = 6.0 microg/ml, where C50 is the plasma concentration associated with a 50% decrease in thrombocytes) and neutrophils (r2 = 0.63; C50 = 0.6 microg/ml). Nolatrexed can be safely administered as an oral preparation at a dose of 800 mg/m2/day for 5 days. Bioavailability was close to 100% and, because inhibition of thymidylate synthase by nolatrexed is rapidly reversible, the slower absorption after a standard meal may result in a shorter duration of noninhibitory concentrations between doses.

Preclinical and phase I clinical studies with the nonclassical antifolate thymidylate synthase inhibitor nolatrexed dihydrochloride given by prolonged administration in patients with solid tumors.

PURPOSE: A phase I, multicenter trial of the thymidylate synthase (TS) inhibitor THYMITAQ (nolatrexed dihydrochloride; Agouron Pharmaceuticals, Inc, San Diego, CA) given by 5-day continuous infusion was performed to establish the maximum-tolerated dose (MTD) and to investigate pharmacokinetics, pharmacodynamics, and antitumor effects. METHODS: In vitro and in vivo preclinical studies demonstrated increased activity with prolonged nolatrexed exposure. In 32 patients, nolatrexed was given as a 5-day infusion at 96 to 1,040 mg/m2/d for 5 days. Pharmacokinetics were determined from high-performance liquid chromatography (HPLC) analyses of plasma and urine. In addition to studying toxicity, plasma deoxyuridine (UdR) elevations were measured as a marker of TS inhibition. RESULTS: The MTD was 904 mg/m2/d for 5 days and the recommended phase II dose is 800 mg/m2/d for 5 days. The dose-limiting toxicity was neutropenia with clinically significant thrombocytopenia and mucositis. These antiproliferative toxicities of nolatrexed were predictable and reversible. A partial response that lasted 3 months occurred in a patient with metastatic colorectal cancer. Pharmacokinetics were nonlinear, with the median plasma clearance (CI) decreasing from 151 mL/min/m2 (range, 124 to 211) at 96 mg/m2/d for 5 days to 49 mL/min/m2 (range, 30 to 84) at 768 mg/ m2/d for 5 days. The half-life (t1/2) was 173 minutes (range, 43 to 784) and 18% (range, 9% to 35%) of the dose was excreted unchanged in the urine. Plasma UdR increased, but returned to pretreatment levels after the end of infusion. Hematologic toxicity was significantly related to nolatrexed plasma concentrations and dose. CONCLUSION: Nolatrexed can be safely administered to patients at a dose of 800 mg/m2/d over 5 days by continuous intravenous infusion and this schedule is associated with antitumor effects. The phase II evaluation of nolatrexed is ongoing.

Clinical pharmacokinetic and pharmacodynamic studies with the nonclassical antifolate thymidylate synthase inhibitor 3, 4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazolone dihydrochloride (AG337) given by 24-hour continuous intravenous infusion.

3,4-Dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazolon e dihydrochloride (AG337) is a nonclassical inhibitor of thymidylate synthase (TS) designed to avoid potential resistance mechanisms that can limit the activity of classical antifolate antimetabolites. A clinical pharmacokinetic and pharmacodynamic study of AG337 given as a 24-h i.v. infusion was performed. Thirteen patients received 27 courses over the dose range 75-1350 mg/m2. Plasma AG337 concentrations were achieved which, in preclinical models, were associated with antitumor effects. AG337 clearance was saturable, and the pharmacokinetics of the drug at doses above 300 mg/m2 was best described by a one-compartment model with saturable elimination (median Km = 6.5 microgram/ml; range, 4.1-13 microgram/ml; median Vmax = 2.0 microgram/ml/h/m2; range, 0.96-5.6 microgram/ml/h/m2). Following the end of the infusion, AG337 was cleared rapidly (t1/2, 53-193 min), and levels were less than 0.2 microgram/ml in all patients by 48 h. Plasma protein binding was 96-98%, and the urinary excretion of AG337 as unchanged drug did not exceed 30% of the dose administered. Measurements of plasma deoxyuridine (dUrd) concentrations showed that doses of 600 mg/m2 and above of AG337 produced a consistent elevation in plasma dUrd levels (60-290%), suggesting that TS inhibition was being achieved in patients. However, in all cases dUrd concentrations had returned to pretreatment levels 24 h after the end of the infusion, suggesting that TS inhibition was not maintained. Local toxicity, probably due to the infusate pH, was the only significant adverse effect observed. These studies have shown that cytotoxic AG337 plasma concentrations can be readily achieved without acute toxicity and that these concentrations are associated with elevations in plasma dUrd levels. The lack of prolonged dUrd elevations indicates that extended administration should be explored using central line or p.o. administration to avoid local toxicity.

In vitro and in vivo antitumor activity of ZENECA ZD0490, a recombinant ricin A-chain immunotoxin for the treatment of colorectal cancer.

ZENECA ZD0490 is a recombinant ricin A-chain-containing immunotoxin that recognizes an antigen that is expressed on approximately 65% of colorectal tumors. The antigen CA242 is recognized by a mouse monoclonal antibody designated C242. C242 antibody was conjugated to recombinant ricin A-chain via a methyl-hindred disulfide linker which confers in vivo stability. ZD0490 was extremely potent against colorectal cell lines CoLo201 and CoLo205, which express the CA242 antigen. ZD0490 activity was determined in vitro by both protein synthesis inhibition (50% inhibitory concentrations of 1-20 ng/ml after 24-h exposure) and clonogenic assay (76-95% cell kill after 24-h exposure to a 50% inhibitory concentration for protein synthesis inhibition; > 99.99% cell kill at 1000 ng/ml). This in vitro activity was translated to in vivo efficacy where single dose i.v. administration of 2.5 mg/kg of ZD0490 was sufficient to induce substantial growth delays of both CoLo201 and CoLo205 s.c. tumors in nude mice. This growth delay equates to between 40 and 60% inhibition of tumor protein synthesis as quantified by an in vivo [14C]leucine incorporation assay. Using this technique, it was shown that protein synthesis inhibition persisted for at least 96 h after a single dose of ZD0490. Administration of the same total dose given as daily doses over 5 days did not alter the antitumor efficacy of ZD0490 in either the growth delay or the protein synthesis inhibition assays. The in vitro and in vivo activity of ZD0490 detailed in this paper show that this novel immunotoxin is worthy of clinical evaluation, which is currently under way in the United Kingdom.

Pharmacokinetic studies in mice with ICI D0490, a novel recombinant ricin A-chain immunotoxin.

A colorectal tumour-directed immunotoxin, ICI D0490, has been constructed by linking recombinant ricin A-chain to C242, a mouse monoclonal antibody, by means of a methyl-hindered disulphide bond. Recombinant ricin A-chain and a hindered disulphide linker were anticipated to confer favourable pharmacokinetic properties on the immunotoxin. The pharmacokinetics of ICI D0490 have been studied in mice following single and repeated i.v. administration. The concentrations of intact immunotoxin in mouse plasma at various time intervals after injection for up to 96 h were measured by a solid-phase enzyme-linked immunosorbent assay (ELISA) and the data analysed by both model-dependent (two compartment) and model-independent methods. Following a single i.v. bolus dose of 2.5 mg kg-1 (50% of the LD10 in mice), the clearance of ICI D0490 from the plasma was extremely slow; 34 microliters min-1 kg-1, t1/2 beta = 33 h. Model-dependent and model-independent analyses gave comparable results with steady state volumes of distribution of 93 and 69 ml kg-1, respectively. The two compartment analysis gave an initial volume of distribution (63 ml kg-1) which is consistent with the predicted plasma volume. Over the dose range 0.05-5 mg ICI D0490 kg-1, plasma levels at 2 and 24 h were linearly related to dose (r > or = 0.98) indicating that at doses up to 5 mg ICI D0490 kg-1 clearance does not appear to have a saturable component. Repeated doses of ICI D0490 (1 mg kg-1 day x 5) did not lead to drug accumulation. These studies demonstrate that ICI D0490 has excellent in vivo stability and persistence which, in conjunction with activity and toxicity data, identify ICI D0490 as a promising candidate for clinical evaluation in the treatment of colorectal cancer.

Genetic heterogeneity of histamine H2-receptors in the mouse vas deferens.

The ability of histamine to inhibit the overall contractile ('twitch') response of the isolated vas deferens of the mouse to electrical field stimulation (64 V pulse, 1 ms pulse width, frequency 0.2 Hz) was studied in nine inbred mouse strains. The strains were also characterized in terms of the potency of the histamine H2-receptor antagonist cimetidine in its inhibition of histamine-mediated effects. An apparently bimodal inter-strain variation (8-10 fold) in both characteristics was encountered, with three strains (SWR, A2G and C57BL/10ScSn) relatively sensitive (S) to both agonist and antagonist actions, and six (C3H, A, C57/BL6, DBA/2, Balb/C and 129/Sv) relatively insensitive (IS). These strain differences were independent of extracellular calcium concentration in the range 1.25-5 mM, and also independent of the frequency of tissue stimulation over the range 0.2-6.4 Hz. Representative S (SWR and A2G) and IS (DBA/2 and C3H) mouse vasa were also characterized in terms of their sensitivity to the agonist actions of dimaprit and the antagonist actions of tiotidine. In the S strain tissues, dimaprit produced 50% inhibition of the twitch response at 4.6-1.8 microM (mean +/- s.d.) and was able to elicit complete inhibition of the twitch response at concentrations greater than 100 microM, whereas 48.7 +/- 11.9 microM dimaprit was required to produce 50% inhibition of the twitch response in tissues from IS mice. In addition, the agonist actions of dimaprit were incomplete in the latter tissues, the drug eliciting no more than 75% inhibition of the twitch response at concentrations in the range 300-1000 microM. Tiotidine produced competitive antagonism of the actions of both histamine and dimaprit, the strain differences being of the same magnitude as those observed for cimetidine. 4 Mating of a representative S (SWR) and IS (129/Sv) strain produced F, mice with intermediate histamine and cimetidine sensitivities relative to the parental strains. A backcross of male F1 to female IS mice produced progeny displaying a range of histamine and cimetidine sensitivities representative of those seen in tissues from F1 and IS parental animals, however, the data were not bimodal. Thus, the backcross data provided no evidence to support single gene inheritance of histamine sensitivity and might suggest that more than one gene is responsible for these differences between S and IS mice.