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 inhibitor, N'°-propargyl-5,8-dideazafolic acid (CB3717). CB3717 was found to be active in early clinical studies, but its use was limited by nephrotoxicity. ICI D1694 is a more potent antitumour agent than CB3717 and is also more water soluble. Previous studies have shown ICI D1694 to be non-toxic to the kidney following a single administration but its renal effects after chronic administration are unknown. To assess these effects, and further define the time course and dose relationship of CB3717-induced renal damage, an assay of glomerular filtration rate (GFR) has been developed which can be used in mice and hence in the screening of novel compounds. The '4C-inulin clearance assay developed was used to show a linear relationship between CB3717 dosage and renal damage (r=-0.989) following a single bolus dose (50-200mgkg-'), in mice. CB3717-induced renal damage is persistent (>6 weeks) and renal scarring was noted. ICI D1694 has been shown to be non-nephrotoxic following weekly administration of 250 mg kg-' for 6 weeks. Measurement of GFR has been shown to be a more sensitive indicator of impaired renal function than plasma urea and creatinine concentration, and the measurement of plasma creatinine concentration in particular, appears to be without value in the screening of potential nephrotoxins in certain mouse strains.Nl'-propargyl-5,8-dideazafolic acid (CB3717, Figure l.i.) is a folate based inhibitor of the enzyme thymidylate synthase (TS), which was found to be an active antitumour agent in early clinical trials in patients with breast, ovarian and hepatocellular carcinoma (Calvert et al., 1986;Cantwell et al., 1988; Bassendine et al., 1987). However, the clinical use of CB3717 was limited by its nephrotoxicity, which was observed in Phase I studies of both weekly (Vest et al., 1988) and 3-weekly administration schedules (Calvert et al., 1986;Sessa et al., 1988). Renal toxicity manifested primarily as a reduction in glomerular filtration rate (GFR). However, tubular damage was also identified by the measurement of the urinary enzymes, N-acetyl glucosaminidase (NAG) and leucine aminopeptidase (LAP). A >20% reduction in GFR was observed in seven of 12 (58%) patients receiving more than 400 mg m2 CB3717. In addition, urinary NAG and LAP levels were elevated in 50% of patients studied, although this elevation was not dose related (Calvert et al., 1986). CB3717-induced nephrotoxicity was thought to be due to the compound's relative insolubility at acid pH. However, despite adequate alkalinisation (pH ,8) of the urine, reductions in GFR (measured using creatinine clearance) of >20%, were still seen in 6/17 (35%) courses in patients treated at 400 mg m2 (Sessa et al., 1988). To circumvent toxicity related to drug precipitation in the kidney, a series of more soluble analogues of CB3717 have been synthesised. In addition to being devoid of renal toxic...

Summary 2-Amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)-quinazoline dihydrochloride (nolatrexed dihydrochloride, Thymitaq, AG337), a specific inhibitor of thymidylate synthase, was developed using protein structure-based drug design. Intravenously administered nolatrexed is active clinically. As oral bioavailability is high (70-100%), nolatrexed was administered orally, 6 hourly for 10 days, at 3-week intervals, and dose escalated from 80 to 572 mg m -2 day -1 in 23 patients. Common toxicity criteria (CTC) grade 3 toxicities included nausea, vomiting, stomatitis and liver function test (LFT) abnormalities. Thrombocytopenia (grade 1 or 2) occurred at doses ≥ 318 mg m -2 day -1 and neutropenia (grade 2) at 429 and 572 mg m -2 day -1 . An erythematous maculopapular rash occurred at dosages ≥ 318 mg m -2 day -1 (7 out of 19 patients). LFT abnormalities occurred in two out of six patients (grade 3 or 4 bilirubin and grade 3 alanine transaminase) at 572 mg m -2 day -1 . Nolatrexed plasma concentrations 1 h after dosing were 6-16 µg ml -1 , and trough 3-8 µg ml -1 , at 572 mg m -2 day -1 . Inhibition of thymidylate synthase was demonstrated by elevation of plasma deoxyuridine. Six-hourly oral nolatrexed for 10 days was associated with antiproliferative effects, but nausea and vomiting was dose limiting at 572 mg m -2 day -1 . Nine patients were treated at 429 mg m -2 day -1 ; three out of nine experienced grade 3 nausea, but 17 out of 22 treatment courses were completed (with the co-administration of prophylactic antiemetics) and this dose level could be considered for phase II testing.

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 inhibitor N10-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 CB3717 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 beta = 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 (greater than or equal to 90% over the range of 20-100 microM). In mice, ICI D1694 CL = 27 ml min-1 kg-1 and t1/2 beta = 30 min following 100 mg/kg i.v., which was significantly faster than CB3717 clearance (CL = 6 ml min-1 kg-1, t1/2 beta = 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 24 h, 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.

Summary 3,5-Dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine (penclomedine, NSC 338720, CRC 88-04) is an a-picoline derivative with anti-tumour activity in preclinical models. Penclomedine administration by 1-h intravenous infusion on 5 consecutive days was repeated 3 weekly in the absence of dose-limiting toxicity (DLT) or disease progression. Five dose levels were investigated (22.5-340 mg m-2 day-'). Eight men and eight women were entered, median age 59 years (range 39-73 years), with good performance status (ECOG 0/1) in 11 patients. A total of 13 out of 16 patients had received previous chemotherapy. Common toxicity criteria grade (CTCg) II vomiting was recorded at all dose levels. Neurotoxicity (cerebellar ataxia and dizziness) was the DLT, CTCg IlIl toxicity occurring in three out of three patients treated at 340 mg m-2 day-'. CTCg IlIl dizziness was noted in one out of three patients at 250 mg m-2 day-1. Neurotoxicity developed during the 1-h infusion and persisted for a variable period (maximum 5 h) after infusion. Prophylactic antiemetic drugs appeared to reduce associated vomiting but did not prevent ataxia. No antiproliferative toxicities were noted and no anti-tumour responses were documented. Penclomedine pharmacokinetic studies confirmed preclinical evidence of extensive apparent distribution (931 m-2) and rapid clearance (41 1 h-1m-2). Purkinje cell loss has been identified in preclinical models after intraperitoneal administration (O'Reilly et al, 1 996a) and further clinical development of penclomedine will focus on oral administration.Keywords: phase l; penclomedine; neurotoxicity Penclomedine (3,5-dichloro-2,4-dimethoxy-6-(trichloromethyl}-pyridine) was originally synthesized by Helen K Tobol, Dow Chemical, USA, as part of a programme to develop effective herbicides. It was identified as a potential anti-tumour agent by the NCI in vivo P388 leukaemia prescreen. After evidence of good activity against murine and human breast tumours, mouse CD8F, mammary carcinoma and human MX-1 mammary tumour xenograft (Plowman et al, 1989), penclomedine was selected for further studies and subsequent clinical development. The mechanism of action of penclomedine is unclear, although studies have been performed that suggest that it undergoes metabolism to yield reactive species that bind to DNA (Plowman et al, 1989; Reid et al, 1992;Benvenuto et al, 1995.) Penclomedine is poorly soluble in water but soluble in nonpolar solvents and lipids (Prankerd et al, 1989). Therefore, an experimental formulation of penclomedine as a 10% oil in water emulsion was developed for intravenous (i.v.) administration. Using this formulation, penclomedine was active against the advanced stage MX-1 mammary carcinoma: oral treatment with doses of 135 mg kg-' day-' for 5 days resulted in ten out of ten tumour-free survivors. The potency increased after intraperitoneal administration and the drug was most potent when administered i.v. (Harrison et al, 1991). No clear schedule dependency was been observed with the oral administratio...

Summary In vivo '9F-NMR spectroscopy has been used to study the pharmacokinetics of the experimental antifolate drug CB3988 (C2-desamino-C2-methyl-N'0-propargyl-2'trifluoromethyl-5,8-dideazafolic acid) in mice and rats. NMR results have been compared to those obtained by HPLC and the effect of the inclusion of the CF3 group evaluated by comparing the pharmacokinetics of CB3988 and ICI 198583 (C2-desamino-C2-methyl-N'°-propargyl-5,8-dideazafolic acid) in rats. In mice, following the administration of CB3988 (500 mg kg-' i.v.), drug could be detected in both the upper and the lower abdomen. NMR signal from the upper abdomen reached maximum intensity 10-40 min after administration, declining thereafter with a half life of 28 min. Signal detected in the lower abdomen reached maximum intensity 60-90 min after treatment. HPLC analyses indicated that CB3988 was present at appreciable concentrations (about 20-30 mg ml-') in both bile and urine which is consistent with the signal from the upper and lower abdomen being derived from the gall bladder and urinary bladder, respectively. Studies in rats also indicated that CB3988 (100 mg kg-' i.v.) rapidly entered and was cleared from the upper abdomen. Comparison of data from rats with intact and cannulated bile ducts suggested that '9F-NMR could detect CB3988 undergoing enterohepatic circulation. Furthermore, comparison of the plasma half life of CB3988 with the half life for the decline of the NMR signal from the upper abdomen suggested that NMR measurements may reflect the plasma clearance of CB3988. When the pharmacokinetics of CB3988 and ICI 198583 were compared the only significant difference was in the alpha phase half life which was 2-fold faster for CB3988. These data demonstrate that CB3988 is cleared rapidly by both biliary and urinary excretion. This is in contrast to N'°-propargyl-5,8-dideazafolic acid, where delayed excretion is associated with hepatic and renal toxicities. The ability to study CB3988 pharmacokinetics non-invasively by '9F-NMR spectroscopy confirms the utility of the technique and, since '9F-NMR can be applied directly to clinical investigations, it may be possible to obtain similar information in humans.