Long-term effects of 7-monohydroxyethylrutoside (monoHER) on DOX-induced cardiotoxicity in mice

Doxorubicin (DOX) is a potent antitumor agent for different types of cancer, but the cumulative, dose-related cardiotoxicity limits its clinical use. The incidence of abnormal cardiac function after treatment with DOX appears to increase with time. Therefore, late cardiotoxicity is—especially in young surviving patients—a major concern. The aim of this study was to evaluate in mice whether the semisynthetic flavonoid 7-monohydroxyethylrutoside (monoHER) also protected against DOX-induced cardiotoxicity after a long period of follow-up. Four groups of 6 Balb/c mice were treated weekly during 6 weeks with saline, DOX alone (4 mg/kg i.v.), DOX preceded by monoHER (500 mg/kg i.p.), or DOX preceded by monoHER followed by long-term weekly monoHER injections during the observation period of 6 months. Half of the mice treated with DOX only developed DOX-induced heart failure and died within 6 months of observation. Two mice co-treated with monoHER showed weight loss and shortness of breath, whereas one mouse was found dead in its cage known with weight loss. The group receiving DOX plus long-term repeated doses of monoHER started to lose weight. Five out of six mice in this group developed shortness of breath and died before the end of the study with symptoms of cardiac failure induced by DOX. Statistical comparison of the histological heart damage between the different experimental groups was not possible, because the animals died at different time-points in the observation period and DOX-induced cardiotoxicity progressed with time. Nevertheless, it was clear that the initial cardioprotective effect of monoHER was not prolonged during the half-year observation period. It was even suggested that addition of repeated doses of monoHER tended to aggravate DOX-induced cardiotoxicity. It cannot be excluded that the dose and frequency of monoHER administration is crucial in obtaining an optimal antioxidant activity without a pro-oxidant activity of monoHER. This work was supported in part by grant VU-97-1525 from the Koningin Wilhelmina Foundation, Amsterdam, The Netherlands.     

The influence of the time interval between monoHER and doxorubicin administration on the protection against doxorubicin-induced cardiotoxicity in mice

Purpose: Despite its well-known cardiotoxicity, the anthracyclin doxorubicin (DOX) continues to be an effective and widely used chemotherapeutic agent. DOX-induced cardiac damage presumably results from the formation of free radicals by DOX. Reactive oxygen species particularly affect the cardiac myocytes because these cells seem to have a relatively poor antioxidant defense system. The semisynthetic flavonoid monohydroxyethylrutoside (monoHER) showed cardioprotection against DOX-induced cardiotoxicity through its radical scavenging and iron chelating properties. Because of the relatively short final half-life of monoHER (about 30 min), it is expected that the time interval between monoHER and DOX might be of influence on the cardioprotective effect of monoHER. Therefore, the aim of the present study was to investigate this possible effect. Methods: Six groups of 6 BALB/c mice were treated with saline, DOX alone or DOX (4 mg/kg i.v.) preceded by monoHER (500 mg/kg i.p.) with an interval of 10, 30, 60 or 120 min. After a 6-week treatment period and additional observation for 2 weeks, the mice were sacrificed. Their cardiac tissues were processed for light microscopy, after which cardiomyocyte damage was evaluated according to Billingham (in Cancer Treat Rep 62(6):865–872, 1978). Microscopic evaluation revealed that treatment with DOX alone induced significant cardiac damage in comparison to the saline control group (P<0.001). Results: The number of damaged cardiomyocytes was 9.6-fold (95% CI 4.4–21.0) higher in mice treated with DOX alone than that in animals of the control group. The ratio of aberrant cardiomyocytes in mice treated with DOX preceded by monoHER and those in mice treated with saline ranged from 1.6 to 2.8 (mean 2.2, 95% CI 1.2–4.1, P=0.019). The mean protective effect by adding monoHER before DOX led to a significant 4.4-fold reduction (P<0.001, 95% CI 2.3–8.2) of abnormal cardiomyocytes. This protective effect did not depend on the time interval between monoHER and DOX administration (P=0.345). Conclusion: The results indicate that in an outpatient clinical setting monoHER may be administered shortly before DOX.This work was supported in part by grant VU-97-1525 from the Koningin Wilhelmina Foundation, Amsterdam, The Netherlands.     

Endurance exercise training preserves cardiac function in rats receiving doxorubicin and the HER-2 inhibitor GW2974

Purpose  To determine if endurance exercise training performed prior to administration of the anticancer drugs DOX and GW2974 would be cardioprotective. Methods  Rats remained sedentary or exercise trained for 10 weeks. Following the exercise or sedentary period, rats were randomly assigned to treatment groups. Rats in sedentary and exercise groups received saline or a combination of 10 mg/kg DOX and 30 mg/kg GW2974. Cardiac function was assessed 2, 5, or 10 days following treatments. Results  Sedentary animals receiving DOX/GW2974 experienced significant cardiac dysfunction. At 2-, 5-, and 10-days post, cardiac function in trained, drug-treated animals was significantly preserved. Additionally, animals exercised prior to DOX/GW2974 injections had significantly lower levels of myocardial lipid peroxidation and caspase-3 and -8 activities compared to their sedentary counterparts. Conclusions  Exercise training protected against the cardiac dysfunction associated with DOX/GW2974 administration and may be related to an inhibition in apoptotic signaling.     

Effect of amrinone on anthracycline-induced lethal and cardiac toxicity in mice and rats

The protective role of amrinone against toxicity of anthracyclines was examined in both mice and rats. These two anthracyclincs were selected since they are characterized by different patterns of toxicity. In contrast to doxorubicin, the 4′-deoxy derivative did not cause delayed mortality. The results of this investigation indicate that amrinone is an effective protective agent against acute lethal events induced by both anthracyclines. However, the inotropic agent did not reduce the delayed mortality produced by doxorubicin. This parallels the apparent lack of prevention of doxorubicin-induced myocardial toxicity in CD rats, as determined by ECG changes and by morphologic alterations following multiple drug administrations. The administration of amrinone did not interfere with the antitumor activity of 4′-deoxy-doxorubicin against C-26 colon tumor.     

Plasma and CSF pharmacokinetics of ganciclovir in nonhuman primates

Purpose: The antiviral nucleoside analogue ganciclovir is a potent inhibitor of replication in herpes viruses and is effective against cytomegalovirus infections in immunocompromised patients. Ganciclovir is also used in cancer gene therapy studies that utilize the herpes simplex virus thymidine kinase gene (HSV-TK). The pharmacokinetics of ganciclovir in adults and children have been described previously but there are no detailed studies of the CNS pharmacology of ganciclovir. We studied the pharmacokinetics of ganciclovir in plasma and CSF in a nonhuman primate model that is highly predictive of the CSF penetration of drugs in humans. Methods: Ganciclovir, 10 mg/kg i.v., was administered over 30 min to three animals. Ganciclovir concentrations in plasma and CSF were measured using reverse-phase HPLC. Results: Peak plasma ganciclovir concentrations ranged from 18.3 to 20.0 μg/ml and the mean plasma AUC was 1075 ± 202 μg/ml · min. Disappearance of ganciclovir from the plasma was biexponential with a distribution half-life (t_1/2α) of 18 ± 7 min and an elimination half-life (t_1/2β) of 109 ± 7 min. Total body clearance (Cl_TB) was 9.4 ± 1.6 ml/min/kg. The mean CSF ganciclovir AUC was 168 ± 83 μg/ml · min and the mean peak CSF concentration was 0.7 ± 0.3 μg/ml. The ratio of the AUCs in CSF and plasma was 15.5 ± 7.1%. Conclusions: Ganciclovir penetrates into the CSF following i.v. administration. This finding will be useful in the design of gene therapy trials involving the HSV-TK gene followed by treatment with ganciclovir in CNS or leptomeningeal tumors. Received: 8 May 1998 / Accepted: 25 September 1998     

Cardiac toxicity events in the PHARE trial,an adjuvant trastuzumab randomised phase III study

BackgroundThis article reports, the cardiac toxicity according to 6- versus 12-month durations of adjuvant trastuzumab in PHARE randomised trial (NCT00381901).Patients and methodsCardiac follow-up and Left Ventricular Ejection Fraction (LVEF) assessment by echocardiography or multigated acquisition scan were performed every 3 months while patients received trastuzumab and after completion of treatment over the first 2 years and every 6 months afterwards. The primary cardiac end-point was Cardiac Heart Failure (CHF) defined as New York Heart Association (NYHA) class III or IV. The secondary cardiac end-points were: cardiac events, cardiac dysfunctions defined by NYHA class I and II; LVEF decreases, cardiac recoveries. The cardiac subcommittee reviewed cardiac events and assessed if patients had favourable outcomes or not on the basis of trends from LVEF measurements.ResultsAmong 3380 patients the cardiac dysfunction assessment included 14,055 and 13,218 LVEF measurements in the 12- and 6-month arms. The overall incidences of CHF were 0.65% (11/1690) and 0.53% (9/1690) in the 12 and 6 month arms, respectively (p > 0.05). Cardiac dysfunction occurred in 5.9% (100/1690) and 3.4% (58/1690) of patients in the 12 and 6 month arms, respectively (p = 0.001). Recoveries were observed for the majority patients and 0.79% (27/3380) of patients experienced an unfavourable cardiac outcome.ConclusionPHARE confirm that the incidence of cardiac end-points remains low and mostly reversible after trastuzumab. Identification at baseline of cardiac risk categories of patients should be of interest to provide an optimal adaptation of adjuvant modalities and a shorter duration might be an option.     

Plasma pharmacokinetics of genistein in mice

Genistein (GEN) has recently generated considerable interest as a potential agent for the prevention and treatment of cancer. The present investigation was undertaken to determine if the concentrations of drug shown to inhibit the growth of human tumor cell lines by 50% in vitro (IC50=2-27 mu g/ml) can be achieved and sustained systemically in mice. We found that GEN plasma levels decreased biexponetially from 64 mu g/ml to 0.55 mu g/ml during the initial 40 min after i.v. injection of a 52 mg/kg dose. Mean half-lives of the two initial disposition phases were 2.5+/-0.4 min and 7.1+/-1.1 min in mice treated with doses of 9-52 mg/kg. Plasma profiles of i.v. GEN exhibited a prominent secondary peak near 78 min followed by a terminal decay phase with a 39.5+/-16.8 min half-life. Although these features are suggestive of enterohepatic cycling, the mean apparent total plasma clearance of GEN (66.5+/-7.3 ml/min/kg) was nevertheless similar to hepatic blood flow. The systemic availability of GEN from a 180 mg/kg p.o. dose, which afforded 1.1 mu g/ml peak plasma concentration, was only 12%. Thus, bolus i.v. and p.o. administration of GEN failed to either achieve or adequately sustain plasma levels of the drug within the target range established by in vitro antitumor studies. Plasma levels resulting from i.p. injection of a 185 mg/kg dose were 5-times greater on average than achieved by the p.o. route. While the plasma concentration exceeded the IC50 values for the majority of human cancer cell lines responsive to GEN for only a short period of time, drug levels remained above 2 mu g/ml, the IC50 of the most sensitive cell lines, for 4 h. Extrapolation from the single dose study suggests that repetitive i.p. injection of at least 200 mg/kg GEN every 8 h will afford continuous systemic exposure to potentially cytostatic concentrations of the drug against these cell lines. This information should facilitate efforts to assess the effectiveness of GEN in appropriate in vivo tumor models.     

Continuous Trastuzumab Therapy in Breast Cancer Patients With Asymptomatic Left Ventricular Dysfunction

Background.

Adjuvant trastuzumab is a highly effective targeted treatment that improves survival for patients with HER2-positive breast cancer. However, trastuzumab interruption is recommended for patients who develop treatment-induced cardiotoxicity (i.e., decline in left ventricular ejection fraction [LVEF], with or without symptoms) and can lead to an incomplete course of treatment. We studied the cardiac safety of continuous trastuzumab therapy among patients with asymptomatic declines in LVEF.

Methods.

We retrospectively evaluated patients with HER2-positive breast cancer treated with adjuvant trastuzumab at our institution between 2005 and 2010. Treatment-induced cardiotoxicity was defined by an absolute decrease in LVEF of ≥10% to below 55% or an absolute decrease of ≥16%. Logistic regression was used to determine the association between candidate risk factors and treatment-induced cardiotoxicity.

Results.

Among 573 patients, 92 (16%) developed treatment-induced cardiotoxicity. Trastuzumab was continued without interruption in 31 of 92 patients with treatment-induced cardiotoxicity—all were asymptomatic with LVEF of ≥50% at cardiotoxicity diagnosis with median LVEF of 53% (range, 50%–63%), and none developed heart failure during follow-up. Risk factors associated with treatment-induced cardiotoxicity included age (p = .011), anthracycline chemotherapy (p = .002), and lower pretrastuzumab LVEF (p < .001).

Conclusion.

Among patients who develop asymptomatic treatment-induced cardiotoxicity with LVEF of ≥50%, continuous trastuzumab therapy appears to be safe.

Implications for Practice:

Cardiotoxicity is the most common reason for patients with HER2-positive breast cancer to receive an incomplete course of life-saving trastuzumab therapy. Data from this study suggest that continuous trastuzumab may be safe in patients with asymptomatic cardiotoxicity and left ventricular ejection fraction of ≥50%. Given the substantial oncologic benefit of trastuzumab, increasing efforts are needed to ensure that patients complete the full course of treatment without interruption. Current recommendations for trastuzumab interruption in patients who develop cardiotoxicity should be re-evaluated.     

Salivary and plasma pharmacokinetics of topotecan in patients with metastatic epithelial ovarian cancer

The comparative saliva/plasma pharmacokinetics of topotecan were investigated in 13 patients with metastatic epithelial ovarian cancer receiving topotecan (30-min intravenous (i.v.) infusion) on a five consecutive day schedule every 3 weeks. During the first and the second courses of treatment, each patient underwent pharmacokinetic evaluation. Quantitation of the total topotecan (lactone plus carboxylate form) was assessed by a highly specific high-performance liquid chromatographic (HPLC) method. Large patient-to-patient variations in the plasma and saliva concentrations were observed. Plasma and saliva pharmacokinetics could be described using a biexponential pattern. From the saliva data, the half-life of the terminal part of the curve was 2.64 h, it was of the same order of magnitude as the topotecan elimination half-life determined from the plasma data, 3.18 h. Topotecan concentrations were higher in the saliva than in the plasma, the saliva/plasma concentration ratio averaged 2.31 and the ratio area under the parotid saliva (AUC_s) over plasma (AUC_p) concentration–time curve (AUC_s/AUC_p) averaged 2.11. For each individual, a significant relationship was found between topotecan concentrations in the saliva and in the plasma, the coefficients of correlation ranged from 0.75 to 0.92 according to the patient. Myelosuppression, especially granulocytopenia was the most frequent toxicity encountered during the trial. The percent decrease in the leucocyte count, absolute neutrophil count and platelet count were related to the AUCp/day using sigmoidal E_max models. The high values of the Hill constant found reflect the very steep AUC-haematoxicity relationship observed. In most cases, abdominal pain occurred in patients presenting high saliva concentrations. One patient with high salivary concentrations (mean S/P RATIO=4.60) had grade 1 mucositis. In conclusion, the concentration of topotecan in saliva appeared to be useful as an indirect, non-invasive estimation of the levels of topotecan in the plasma; thus, saliva concentrations could be a good predictor of the behaviour of topotecan in the body.     

Plasma and cerebrospinal fluid pharmacokinetics of rebeccamycin (NSC 655649) in nonhuman primates

Purpose The rebeccamycins, indolocarbazole topoisomerase I poisons originally discovered in actinomycetes, have shown activity in vitro against a range of adult and pediatric tumors. The derivative NSC 655649 (diethylaminoethyl analog of rebeccamycin, or DEAE rebeccamycin) is currently undergoing early-phase human studies and has shown some signs of antitumor activity. We studied the plasma and cerebrospinal fluid (CSF) pharmacokinetics of NSC 655649 after systemic administration in a nonhuman primate model that is predictive of anticancer drug behavior in humans.Design A dose of 400 mg/m² was infused over 1 h to three rhesus monkeys. Serial blood and CSF samples were collected. Rebeccamycin concentrations were measured by high-pressure liquid chromatography. Pharmacokinetic analysis was performed using compartmental and noncompartmental methods.Results A two-compartment or three-compartment model described rebeccamycin pharmacokinetics in plasma adequately. In two animals, the three-compartment model provided a better fit, and in one animal, the two-compartment model was better. The terminal half-life was 730±410 min, the AUC was 3130±425 M min, and the clearance was 190±25 ml/min/m². Rebeccamycin was below the limit of quantitation in all CSF samples. The animals had some nausea and agitation during and shortly after the infusion that responded to treatment with prochlorperazine or diazepam. Otherwise, rebeccamycin was well tolerated with minimal toxicity.Conclusion Rebeccamycin penetrates poorly into the CSF following an intravenous infusion. Therefore, systemically administered rebeccamycin is unlikely to be an important agent for the treatment of leptomeningeal tumors. Because the drug is associated with local irritation at injection sites, it is not an ideal candidate for development as an intrathecal agent. However, the role of rebeccamycin in the treatment of parenchymal brain tumors should be determined in clinical trials.     

Bioavailability and pharmacokinetics of oral topotecan: a new topoisomerase I inhibitor.

The results of preclinical and clinical studies indicate enhanced antineoplastic activity of topotecan (SKF 104864-A) when administered as a chronic treatment. We determined the apparent bioavailability and pharmacokinetics of topotecan administered orally to 12 patients with solid tumours in a two-part crossover study. The oral dose of 1.5 mg m-2 was administered as a drinking solution of 200 ml on day 1. The i.v. dose of 1.5 mg m-2 was administered as a 30 min continuous infusion on day 2. The bioavailability was calculated as the ratio of the oral to i.v. area under the curve (AUC) calculated up to the last measured time point. The oral drinking solution was well tolerated. The bioavailability revealed moderate inter-patient variation and was 30% +/- 7.7% (range 21-45%). The time to maximum plasma concentration after oral administration (Tmax) was 0.78 h (median; range 0.33-2.5). Total i.v. plasma clearance of topotecan was 824 +/- 154 ml min-1 (range 535-1068 ml min(-1)). The AUC ratio of topotecan and the lactone ring-opened hydrolysis product (hydroxy acid) was of the same order after oral (0.34-1.13) and i.v. (0.47-0.98) administration. The bioavailability of topotecan after oral administration illustrates significant systemic exposure to the drug which may enable chronic oral treatment.     

Dose-dependent pharmacokinetics of methotrexate and 7-hydroxymethotrexate in the rat in vivo

The pharmacokinetics of methotrexate (MTX) and 7-hydroxymethotrexate (7-OH-MTX) in bile, urine, and serum was studied in rats in vivo after short-time infusions of 10, 50, 250, and 1000 mg/kg MTX. All animals were anesthetized and drained of bile during experiments. The biliary secretion rate of MTX approached saturation when serum MTX levels surpassed 700-800 microM, causing a significant reduction in biliary recovery as the parent compound (49 to 32%) at MTX doses exceeding 50 mg/kg. The hepatic metabolism of MTX to the 7-hydroxy metabolite was not saturated at the doses used. Serum MTX pharmacokinetics demonstrated dose dependency, inasmuch as doses exceeding 10 mg/kg were accompanied by a reduced total body clearance (Clr) and biliary clearance (ClB). A significant finding in relation to acute hepatotoxicity reported after high-dose MTX in humans was the occurrence of cholestasis 30-90 min after drug infusion and the observation of macroscopic precipitations in the bile duct in five of six animals treated with 1000 mg/kg MTX. In these five animals, cessation of bile secretion occurred at similar bile 7-OH-MTX levels [9800 +/- 1100 (SD) microM], while the single rat that secreted bile throughout the experiment had a 5-fold lower peak 7-OH-MTX concentration in bile. Analysis of biliary precipitates formed in vivo and in vitro found 7-OH-MTX to constitute 97% and MTX 3% of the drug content of the precipitated material.     

Minimizing Cardiotoxicity While Optimizing Treatment Efficacy with Trastuzumab: Review and Expert Recommendations

Numerous clinical studies have demonstrated the therapeutic benefit of trastuzumab in women with breast cancer. However, a small but not insignificant proportion of patients have experienced trastuzumab‐associated cardiotoxicity during these trials. This phenomenon is generally characterized by an asymptomatic reduction in left ventricular ejection fraction (LVEF) or, less often, congestive heart failure (CHF). Concomitant anthracycline therapy significantly increases the risk for cardiotoxicity during trastuzumab treatment, and such regimens are therefore not recommended. The cardiac dysfunction associated with trastuzumab is most often reversible upon discontinuation of treatment and initiation of standard medical therapy for CHF. Prior to treatment initiation, a risk–benefit analysis should be performed for each individual patient, including a thorough assessment of potential risk factors and cardiac function. Cardiac monitoring should be continued throughout trastuzumab therapy and the follow‐up period, because early recognition of trastuzumab‐associated cardiac dysfunction can allow effective medical intervention. Following the occurrence of asymptomatic LVEF reduction or CHF and appropriate medical intervention, reintroduction of trastuzumab may be considered in patients following resolution of normal cardiac function, or in those for whom the benefit of antitumor therapy outweighs the risk for CHF.     

A phase I study of monohydroxyethylrutoside in healthy volunteers

The flavonol monohydroxyethylrutoside (monoHER) has demonstrated protection against doxorubicin-induced cardiotoxicity in in vitro and in vivo studies without affecting the antitumor effect. In the present phase I study, the possible side effects and the pharmacokinetics of monoHER were evaluated in healthy volunteers with the aim to develop a safe and feasible dose to be evaluated in cancer patients treated with doxorubicin. The study was performed as a single blind, randomized trial in healthy volunteers (age between 19 and 56 years). At each dose level, six subjects received monoHER and three placebo. MonoHER was solubilized in 100 ml dextrose 5% and administered as an i.v. infusion in 10 min. The placebo consisted of 100 ml dextrose 5%. The starting dose of monoHER was 100 mg/m². Dose escalation by 100% of the preceding dose took place after finishing each dose level until the protecting pharmacokinetic values for C _max and AUC^∞ (as observed in mice after 500 mg/kg monoHER i.p.) were reached and/or serious side effects were observed. The dose was escalated up to 1,500 mg/m². The mean values of C _max and AUC^∞ were 360±69.3 μM and 6.8±2.1 μmol min/ml, respectively. These values were comparable to the C _max and AUC^∞ observed under the protecting conditions in mice. No serious side effects occurred during the entire study. Thus, 1,500 mg/m² is a feasible and safe dose to be evaluated in a phase II study to investigate the protective properties of monoHER against doxorubicin-induced cardiotoxicity in cancer patients. Preliminary results of this study have been presented as a poster at AACR (Proc. AACR 43 (2002) 2751).     

Influence of piperacillin on the pharmacokinetics of methotrexate and 7-hydroxymethotrexate

The influence of concomitant administration of piperacillin (PIP) on the pharmacokinetic parameters of methotrexate (MTX) and 7-hydroxymethotrexate (7-OH-MTX) was studied in rabbits. Six rabbits received an initial i.v. bolus (0.21 mg kg⁻¹) followed by a constant-rate i.v. infusion of the drug (5 μg min⁻¹ kg⁻¹) for 240 min. The PIP dose (30 mg kg⁻¹) was repeated every 30 min until the end of the infusion period. The control group consisted of four rabbits treated the same way except for the addition of PIP. There were significant increases in the mean residence times found for MTX (MRT_inf) and 7-OH-MTX (MRT_m,inf) following PIP administration. Concomitant administration of PIP with MTX also produced significant 1.5- and 2.8-fold increases in the area under the curve of MTX and 7-OH-MTX, respectively. The total body clearance of MTX and the operative total body clearance of 7-OH-MTX significantly decreased, but in a less than proportional manner. The study demonstrates that the interaction between MTX and PIP is mainly due to the reduced clearance of both MTX and 7-OH-MTX combined with a slight increase in the formation clearance of the metabolite. Received: 9 March 1998 / Accepted: 14 May 1998     

The pharmacokinetics of the quinazoline antifolate ICID 1694 in mice and rats

Summary N-(5-[N-(3,4-Dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-l-glutamic acid (ICI D1694) is an analogue of the thymidylate synthase inhibitorN ¹⁰-propargyl-5,8-dideazafolic acid (CB3717). CB3717 was found to be an active anticancer agent in early clinical studies, but its use was limited by its relative insolubility at physiological pH. ICI D1694 has been shown to be a more active anticancer agent than CB 3717 in model systems, and it is devoid of the acute renal toxicity associated with the administration of the latter drug to mice. In the present study, the pharmacokinetics of ICI D1694 were studied in both mice and rats using reverse-phase HPLC. In rats, ICI D1694 clearance (CL) conformed to a two-compartment open model and was rapid (CL=10.7 ml min^–1 kg^–1,t1/2=30 min). Excretion was mainly biliary (65% of the delivered dose in 4 h vs 12% in urine) in the rat following a 100-mg/kg i.v. bolus. A high degree of protein binding was seen in rat plasma (90% over the range of 20–100 m). In mice, ICI D1694CL=27 ml min^–1 kg^–1 andt1/2=30 min following 100 mg/kg i.v., which was significantly faster than CB3717 clearance (CL=6 ml min^–1 kg^–1,t1/2=93 min). ICI D1694 was fully bioavailable following i.p. administration (AUC=3.73 mg ml^–1 min i.v. 4.03 mg ml^–1 min i.p.), but its bioavailability following oral administration appeared to be low (approximately 10%–20%). Tissue distribution and excretion studies in mice suggested that biliary excretion predominated, confirming the results obtained in rats. Following an i.v. dose of 500 mg/kg ICI D1694 in mice, drug was detectable at 24h, suggesting the presence of a third phase of plasma clearance. The initial HPLC assay could not detect this third phase following a dose of 100 mg/kg; hence, a more sensitive assay was developed that includes a solid-phase extraction step. The latter assay was used to define the third phase of ICI D1694 clearance in mice, and preliminary studies demonstrated a terminal half-life of 6.5±2.7 h.These studies were supported by the UK Cancer Research Campaign and the British Technology Group