Safety and Toxicity of Recombinant Methioninase and Polyethylene Glycol (PEG) Recombinant Methioninase in Primates.

Methionine (MET) is a general metabolic therapeutic target in cancer, whereby cancer cells have an elevated requirement for MET, termed MET dependence. We have developed recombinant L-methionine α-deamino-γ-mercaptomethane lyase (recombinant methioninase [rMETase, EC 4.4.1.11]) as targeted therapy of all cancer types. Pharmacokinetics, MET depletion, antigenicity, and toxicity of rMETase were examined in macaque monkeys. Pharmacokinetic analysis showed that rMETase was eliminated with a T1/2 of 2.49 h. A 2-week i.v. administration of 4000 units/kg every 8 h/day for 2 weeks resulted in a steady-state depletion of plasma MET to less than 2 µM. The only manifest toxicity was decreased food intake and slight weight loss. Serum albumin and red-cell values declined transiently during treatment. Rechallenge on day 28 resulted in anaphylactic shock and death in one animal. Pretreatment with hydrocortisone prevented the anaphylactic reaction. Anti-rMETase antibodies (at 10-3) were found after the first challenge, increased to 10-6 after the fourth challenge, and decreased to 10-2 by 2 months post-therapy. Therefore, the therapeutic potential of rMETase is limited by its short plasma half-life and immunologic effects, including high antibody production in mice and anaphylactic reactions in monkeys. To overcome these limits, rMETase has been coupled to methoxypolyethylene glycol succinimidyl glutarate polyethylene glycol (MEGC-PEG-5000). The pharmacokinetics, antigenicity, and toxicity of MEGC-PEG-rMETase in macaque monkeys were evaluated using an escalating-dose strategy. In pharmacokinetic studies, a single 4000 units/kg dose showed that MEGC-PEG-rMETase holoenzyme activity was eliminated with a biological half-life of 1.3 h, and the MEGC-PEG-rMETase apoenzyme was eliminated with a biological half-life of 90 h, a 36-fold increase compared with non-PEGylated rMETase. The disparity in the T½ of the apoenzyme and the holoenzyme reflects the loss of co-factor pyridoxal-L-phosphate of the circulating MEGC-PEG-rMETase. A 7-day i.v. administration of 4000 units/kg every 12 h resulted in a steady-state depletion of plasma MET to <5 µmol/L. The only manifest toxicity was decreased food intake and slight weight loss. Red cell values and hemoglobin declined transiently. Subsequent challenges did not result in any immunologic reactions. Anti-MEGC-PEG-rMETase antibodies were 100- to 1000-fold less than antibodies elicited by naked rMETase, thereby suggesting clinical potential of MEGC-PEG-rMETase as a broad anticancer agent.

Serum methionine depletion without side effects by methioninase in metastatic breast cancer patients.

The growth dependence of human tumors on elevated levels of methionine has been shown in preclinical in vitro and in vivo studies to be a frequently occurring, highly effective, tumor-selective therapeutic target. High purity endotoxin-free methioninase was produced from Pseudomonas putida in order to develop anti-methionine chemotherapy targeting of human tumors. A pilot Phase I clinical trial has been initiated in order to determine methioninase toxicity, the pharmacokinetics of methioninase and methionine-depletion and maximum tolerated dose. A two hour i.v. infusion of 5,000 units (0.4 g) and 10,000 units (0.8 g) and a ten hour i.v. infusion of 20,000 units (1.6 g) of methioninase was administered to patient-1, patient-2, and patient-3, respectively. All patients had advanced breast cancer. Blood and urine samples were obtained at frequent intervals between 0 and 24 hours. The toxicity evaluations were carried out according to FDA criteria. Pharmacokinetics data were obtained for both methioninase and methionine levels in the serum. No acute clinical toxicity was observed for all the toxicity criteria measured in patient-1, patient-2 and patient-3. The depletion of serum methionine started within 30 minutes of the infusion, and was maintained for 4 hours after the infusion was completed in patient-1 and patient-2. The lowest serum methionine levels were 35% and 19% of the pretreatment level, respectively, in patient-1 and patient-2. Patient-3 received a ten hour i.v. infusion of 20,000 units of methioninase without any signs of side effects. Patient-3 maintained serum levels of methioninase as high as 50% of the maximum level for a subsequent 6 hours after infusion. Methionine was depleted over 200-fold from 23.1 microM to 0.1 microM by the 10-hour infusion of patient-3. No clinical toxicity was observed whatsoever in all the toxicity criteria measured in patient-3. The results of the methioninase pilot Phase 1 clinical trial suggested that i.v. infusion of the methioninase is safe and effectively depletes serum methionine without any signs of side effects. Clinical studies are continuing to determine the maximum length of time complete serum methionine depletion can be tolerated.