Seventy-four previously untreated patients with metastatic colorectal adenocarcinoma were prospectively randomized into one of three treatment regimens: (1) 5-fluorouracil (5-FU) 450 mg/m2 as an intravenous (IV) bolus daily for five days or toxicity, then 200 mg/m2 IV bolus every other day for six doses; (2) methotrexate (MTX) 50 mg/m2 in normal saline by IV infusion over four hours followed by an IV bolus of 5-FU 600 mg/m2. This was administered weekly for 4 weeks and then every 2 weeks. (3) Leucovorin 500 mg/m2 in a two-hour IV infusion of normal saline with 5-FU 600 mg/m2 as an IV bolus one hour after the Leucovorin began every week for 6 weeks. The combined complete and partial response rates in the three regimens were 11%, 5%, and 48%, respectively (P = .0009). The median duration of response in the 5-FU and Leucovorin regimen was 10 months. There was no statistically significant difference between the treatment regimens with respect to survival time (P = .6). Toxicity in the 5-FU and Leucovorin regimen was predominantly diarrhea (13 of 30 patients, 40%). In this regimen, eight of 13 patients (52%) who developed diarrhea not only required a dose reduction of 5-FU, but also hospitalization for IV hydration. The predominant toxicity in the 5-FU alone regimen and the 5-FU and MTX regimen was leukopenia. One drug-related death occurred in each regimen.

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Summary Studies with a conjugate of carboxypeptidase G2 (CPG2) and the F(ab')2 fragment of monoclonal anti-CEA antibody, A5B7, have shown specific localisation in a human colon tumour xenograft, LS174T, growing in nude mice. The conjugate reaches a peak concentration in the tumour within 24 h but enzyme activity in blood remains above a critical value for therapeutic purposes for several days. Here we describe a new monoclonal antibody, SB43, raised against CPG2 which is capable of reducing enzyme activity in blood. In vitro studies demonstrated specific binding of SB43 to CPG2 causing inactivation. Moreover, in the nude mouse model SB43 was also capable of inactivating the enzyme in the circulation within minutes of administration. Radiolabelled native SB43 persisted in blood for several days and appreciable non-specific uptake into the xenograft was also observed. Uptake of SB43 by the tumour, with possible inactivation of CPG2 at this site, could be limited by first coupling the antibody to galactose. This ensured recognition and excretion of SB43 and SB43-enzyme complexes via the liver and their rapid removal from the circulation. Galactosylation had no effect on the ability of SB43 to inactivate the enzyme.Anti-cancer agents such as drugs, toxins and radioisotopes have been linked directly to antibodies to achieve greater anti-tumour selectivity but this approach has so far proved to be of restricted therapeutic value. Major limitations resulting from the use of directly coupled antibodies are the amount of anti-cancer agent that can be attached to the antibody molecule without loss of immunological activity and the amount of conjugate that can be specifically targeted to tumour. A novel approach of converting a prodrug into a potent anticancer agent by a targeted enzyme (ADEPT) aims to overcome these difficulties. As originally envisaged (Bagshawe, 1987) this system employed two phases in which an enzyme conjugated to an anti-tumour antibody was first allowed to localise and clear from the circulation before injection of the prodrug. Our preliminary studies (Bagshawe et al., 1988) were carried out with carboxypeptidase G2 (CPG2), a folate depleting bacterial enzyme, conjugated to the F(ab')2 fragment of a monoclonal anti-hCG antibody and capable of converting a glutamyl benzoic acid mustard prodrug into a potent benzoic acid mustard. In the choriocarcinoma xenograft system tested, the target antigen, hCG, was present in the plasma and clearance of the antibody-enzyme conjugate from the circulation occurred within 72 h allowing administration of the prodrug and elimination of the tumour. A similar approach, in which placental alkaline phosphatase was used to convert the prodrug etoposide phosphate into etoposide, was shown to be effective in a colon xenograft model (Senter et al., 1988). We have now extended our studies to the human colonic carcinoma xenograft model (LS174T) in which we have targeted CPG2 conjugated to the monoclonal antibody fragment, A5B7-F(ab')2, directed at carcinoembryonic antigen (CEA...

Sunmary A novel therapy for improving selectivity in cancer chemotherapy aims to modify distribution of a cytotoxic drug by generating it selectively at tumour sites. In this approach an antibody-enzyme conjugate is allowed to localise at the tumour sites before injecting a prodrug which is converted to an active drug specifically by the targeted enzyme in the conjugate. We present here pharmacokinetic studies on the prodrug 4-(bis (2-chloroethyl) amino) benzoyl-L-glutamic acid and its activated derivative, benzoic acid mustard. The glutamic acid is cleaved from the prodrug to form the active drug by carboxypeptidase G2 (CPG2), an enzyme from Pseudomonas sp., which is not found in mammalian cells. The prodrug and its parent active drug were rapidly distributed in plasma and tissues after administration of prodrug or active drug (41 tsmol kg-' intraperitoneally) to mice bearing human choriocarcinoma xenografts. Prodrug and active drug both followed a two-compartment kinetic model. Prodrug was eliminated more rapidly (t,/2 = 0.12 h, t,/2P = 0.70 h) than active drug (t,/2a = 0.37 h, t,12P = 1.61 h). Conversion of the prodrug to the activated parent drug was detected within 5 min of administration to mice which had previously received a F(ab')2-anti-human chorionic gonadotrophin antibody (W14A) conjugated to the enzyme, CPG2 (1,000 U kg-'). Tumour was the only tissue that activated all the prodrug reaching the site. It contained the highest concentration of targeted enzyme conjugate capable of catalysing the reaction of prodrug to drug. Plasma and other tissues were also capable of activating the prodrug but active drug production was limited by the amount of enzyme present. The active drug measured in plasma and tissues other than tumour was attributable to residual antibody-enzyme conjugate at non-tumour sites. Low levels of conjugate in tissues and plasma militate against the advantage of tumour localised enzyme therefore necessitating removal of non-localised enzyme.

Indiplon, at doses of 10 mg in adults and 5 mg in the elderly, was not associated with next day residual sedation or impairment in simple cognitive and psychomotor tasks when administered during the night 4 h prior to awakening.