The use of glucarpidase as a rescue therapy for high dose methotrexate toxicity - a review of pharmacological and clinical data.

INTRODUCTION: This review aims to summarize recent data on the pharmacodynamic, pharmacokinetic, and safety of glucarpidase. This is an enzymatic agent that catalyzes the conversion of methotrexate (MTX) into inactive metabolites. Glucarpidase is used to manage high-dose MTX (HDMTX) toxic plasma concentration, especially in patients with impaired renal function. AREAS COVERED: In this review, studies on glucarpidase clinical efficacy as a therapeutic option for patients suffering from MTX kidney toxicity were presented. Pharmacodynamic and pharmacokinetic properties of glucarpidase were included. Moreover, potential interactions and safety issues were discussed. EXPERT OPINION: The use of glucarpidase is an effective therapeutic strategy in both adults and children treated with high doses of MTX for various types of cancer who have developed acute renal failure. Glucarpidase causes MTX to be converted to nontoxic metabolites and accelerates the time for its complete elimination. After administration of glucarpidase, it is possible to resume HDMTX.

Massively parallel, computationally guided design of a proenzyme.

Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by ∼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.

TAT-mediated intracellular delivery of carboxypeptidase G2 protects against methotrexate-induced cell death in HepG2 cells.

Side effects of methotrexate (MTX) especially hepatotoxicity limits clinical applications of this anticancer agent. Carboxypeptidase G2 (CPG2) is administrated for the treatment of elevated plasma concentrations of MTX. In this study, we have investigated the intracellular delivery of CPG2 fused to the transactivator transduction domain (TAT) and its protective effects against MTX-induced cell death of HepG2 cells. We have observed that both native and denatured forms of the enzyme transduced into the HepG2 cells efficiently in a concentration and time-dependent manner. The denatured protein transduced with higher efficiency than the native form and was functional inside the cells. MTX exposure significantly decreased HepG2 cell viability in a dose- and time-dependent manner. The cell viability after 24 and 48 h of incubation with 100 µM MTX was reduced to 44.37% and 17.69%, respectively. In cells pretreated with native and denatured TAT-CPG2 protein the cell viability was 98.63% and 86.31% after 24 and 48 h, respectively. Treatment with MTX increased the number of apoptotic HepG2 cells to 90.23% after 48 h. However, the apoptosis percentage in cells pretreated with native and denatured TAT-CPG2 was 21.49% and 22.28%, respectively. Our results showed that TAT-CPG2 significantly prevents MTX-induced oxidative stress by decreasing the formation of ROS and increasing the content of glutathione (GSH) and catalase activity. Our finding indicates that both native and denatured TAT-CPG2 strongly protect HepG2 cells against MTX-induced oxidative stress and apoptosis. Hence, intracellular delivery of CPG2 might provide a new therapeutic strategy for protecting against MTX mediated cytotoxicity.

Glucarpidase for the treatment of life-threatening methotrexate overdose.

High-dose methotrexate (HDMTX) is widely and safely used in oncology, with adequate measures including vigorous hydration, urine alkalinization and leucovorin rescue. Despite these precautions, some patients still develop HDMTX-induced nephrotoxicity, which leads to delayed methotrexate (MTX) clearance and sustained elevated plasma MTX levels, which can significantly increase MTX toxicity. Glucarpidase (carboxypeptidase G2, Voraxase®) is a recombinant bacterial enzyme that rapidly hydrolyzes MTX to inactive metabolites, providing an alternate non-renal pathway for MTX elimination in patients with renal dysfunction during HDMTX treatment. Glucarpidase has recently been approved for the treatment of toxic plasma MTX concentrations in patients with delayed MTX clearance due to impaired renal function. Preclinical and clinical studies demonstrated good safety and efficacy in rapidly reducing elevated MTX levels. Further comparative studies are awaited to confirm the benefit of glucarpidase in terms of toxicity and survival.

A novel vascular endothelial growth factor-directed therapy that selectively activates cytotoxic prodrugs.

We have generated fusion proteins between vascular endothelial growth factor (VEGF) and the bacterial enzyme carboxypeptidase G2 (CPG2) that can activate the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA). Three asparagine residues of CPG2 were mutated to glutamine (CPG2(Q)3) to prevent glycosylation during secretion, and truncations of VEGF(165) were fused to either the C- or N-terminal of CPG2. The K(m) of the fusion proteins (37.5 microM) was similar to that of secreted CPG2(Q)3 (29.5 microM) but greater than that of wild-type CPG2 (8 microM). The affinity of the fusion proteins for VEGF receptor-2 (VEGFR2) (K(d)=0.5-1.1 nM) was similar to that of [(125)I]VEGF (K(d)=0.5 nM) (ELISA) or slightly higher (K(d)=1.3-9.6 nM) (competitive RIA). One protein, VEGF(115)-CPG2(Q)3-H(6), possessed 140% of the enzymic activity of secreted CPG2(Q)3, and had a faster half-maximal binding time for VEGFR2 (77 s), than the other candidates (330 s). In vitro, VEGF(115)-CPG2(Q)3-H(6) targeted CMDA cytotoxicity only towards VEGFR-expressing cells. The plasma half-life of VEGF(115)-CPG2(Q)3-H(6) in vivo was 3 h, comparable to equivalent values observed in ADEPT. We conclude that enzyme prodrug therapy using VEGF as a targeting moiety represents a promising novel antitumour therapy, with VEGF(115)-CPG2(Q)3-H(6) being a lead candidate.

Induction of apoptosis by the ADEPT agent ZD2767: comparison with the classical nitrogen mustard chlorambucil and a monofunctional ZD2767 analogue.

ZD2767P is a phenol mustard glutamate prodrug which is currently being developed for Antibody Directed Enzyme Prodrug Therapy (ADEPT). In ZD2767 ADEPT an active bi-functional alkylating drug, ZD2767D (4-[N,N-bis(2-iodoethyl)amino]phenol), is generated following cleavage of ZD2767P by the bacterial enzyme carboxypeptidase G2 (CPG2) which is targeted to the tumour by conjugation to the F(ab')(2)fragment of the anti-CEA antibody A5B7. The aim of the studies described here was to identify the mode of cell death induced by ZD2767P + CPG2 in comparison to the established nitrogen mustard chlorambucil. The contribution of bifunctional and monofunctional ZD2767 DNA lesions to cell death induction was investigated using a monofunctional ZD2767D analogue. Apoptosis in LoVo tumour cells was studied by three different methods (nuclear morphology, annexin V staining and TUNEL). Levels of apoptosis detected using the three assays were similar, and each drug treatment produced apoptosis at levels above those in control cells at concentrations which resulted in tumour cell growth inhibition. The bi-functional compounds, ZD2767P + CPG2 and chlorambucil, induced apoptosis in a concentration and time dependent manner, with equitoxic concentrations producing equivalent levels of apoptosis. In contrast, the mono-functional ZD2767D analogue at 100 microM resulted in the maximal level of apoptosis at 25 h with no further increase over the following 72 h. These studies have demonstrated that apoptosis is the mechanism of cell death induced by the ZD2767 ADEPT system, and that levels of apoptosis produced by ZD2767 are similar to those observed at equitoxic concentrations of the classical nitrogen mustard chlorambucil. The mono-functional ZD2767 analogue also induced apoptosis, but with a different time course and characteristics. In conjunction with previous data, these studies have shown that the potent activity of ZD2767 can be attributed to the ability of the compound to induce bifunctional DNA lesions and engage apoptosis.

Glutamyl hydrolase. pharmacological role and enzymatic characterization.

gamma-Glutamyl hydrolase (GH, EC 3.4.19.9) is a lysosomal and secreted glycoprotein that hydrolyzes the gamma-glutamyl tail of antifolate and folate polyglutamates. Tumor cells that have high levels of GH are inherently resistant to classical antifolates, and further resistance can be acquired by elevations in GH following exposure to this class of antitumor agents. The highest level of expression in normal tissues occurs in the liver and kidney in humans. When panels of tumors are compared with normal tissues, GH expression is elevated in cancerous hepatic and breast tissue. A second poly-gamma-glutamate hydrolyzing enzyme, glutamate carboxypeptidase II, is a transmembrane protein whose active site is on the outside of the cell, occurring in the prostate gland, small intestine, brain, kidney, and tumor neovasculature. It is a high-affinity (nanomolar), low-turnover, zinc co-catalytic enzyme. In contrast, GH is a low-affinity (micromolar), high-turnover enzyme that has a cysteine at the active site. Data are presented suggesting that Cys110 is the nucleophile that attacks the gamma-amide linkage and causes hydrolysis. GH is being evaluated as an intracellular target for inhibition in order to enhance the therapeutic activity of antifolates and fluorouracil.

Glutamyl hydrolase: properties and pharmacologic impact.

Glutamyl hydrolase cleaves the poly-gamma-glutamate chain folate and antifolate poly-gamma-glutamates. Its cellular location is lysosomal with large amounts of the enzyme constitutively secreted. The highest levels of glutamyl hydrolase mRNA in humans is found in the liver and kidney. Baculovirus-expressed human enzyme has been used to evaluate the method of hydrolysis of methotrexate-gamma-glu4 and MTA-gamma-glu4. In both cases, the substrates are hydrolyzed by removal of the outer two gamma-glutamate linkages, yielding glu and gamma-glu2 as the glutamate products. Cell lines resistant to 5,10-dideazatetrahydrofolate (lometrexol) have sevenfold higher activities of glutamyl hydrolase. These cultures have a 60% to 90% reduced amount of antifolate polygamma-glutamates and 30% reduced folyl poly-gamma-glutamates. These results suggest the possibility of using glutamyl hydrolase to favorably modulate the activity of antifolate therapy.

Novel inhibitors of carboxypeptidase G2 (CPG2): potential use in antibody-directed enzyme prodrug therapy.

The design and synthesis of potent thiocarbamate inhibitors for carboxypeptidase G2 are described. The best thiocarbamate inhibitor N-(p-methoxybenzenethiocarbonyl)amino-L-glutamic acid 6d, chosen for preliminary investigations of in vitro antibody-directed enzyme prodrug therapy (ADEPT), abrogated the cytotoxicity of a combination of A5B7-carboxypeptidase G2 conjugate and prodrug PGP (N-p-{N,N-bis (2-chloroethyl)amino}phenoxycarbonyl-L-glutamate) toward LS174T cells. This is the first report of a small-molecule enzyme inhibitor proposed for use in conjunction with the ADEPT approach.

Trienzyme treatment for food folate analysis: optimal pH and incubation time for alpha-amylase and protease treatment.

Recent reports have indicated that trienzyme treatment before folate determination is essential to obtain the proper folate content in foods. Trienzyme treatment is performed by using alpha-amylase and protease for folate extraction from carbohydrate and protein matrices, and folate conjugase for the hydrolysis of polyglutamyl folates. We evaluated the conditions of pH and incubation time for the treatment with alpha-amylase and protease. Four food items, including fresh beef, white bread, cow's milk, and fresh spinach, were selected for this investigation. We found that optimal pHs for alpha-amylase treatment of beef and cow's milk were 7.0 and 5.0, respectively, whereas those for white bread and spinach were not distinctive at pHs from 2.0 to 7.0. The optimal incubation time for alpha-amylase was 4 h for fresh beef and cow's milk, whereas no distinctive optimal incubation period was found for white bread and fresh spinach. Our data indicate that the conditions for enzyme treatments vary depending on food items. Trienzyme treatment resulted in an increase of more than 50% in the mean folate content over folate conjugase treatment alone. It is necessary to treat food samples with not only traditional folate conjugase, but also with alpha-amylase and protease before folate determination to obtain the actual folate content.

A cell surface tethered enzyme improves efficiency in gene-directed enzyme prodrug therapy.

The potential for expressing the bacterial enzyme carboxypeptidase G2 (CPG2) tethered to the outer surface of mammalian cells was examined for use in gene-directed enzyme prodrug therapy. The affinity of CPG2 for the substrate methotrexate was unaffected by three mutations required to prevent N-linked glycosylation. Breast carcinoma MDA MB 361 cells expressing CPG2 internally showed only a very modest increase in sensitivity to the prodrug CMDA because the prodrug did not enter the cells. Cells expressing surface-tethered CPG2, however, became 16-24-fold more sensitive to CMDA and could mount a good bystander effect. Systemic administration of CMDA to mice bearing established xenografts of the transfected cells led to sustained tumor regressions or cures.

Carboxypeptidase G2 rescue after high-dose methotrexate.

PURPOSE: This study was a pilot project to assess the safety and efficacy of carboxypeptidase G2 (CPG2) rescue from high-dose (HD) methotrexate (MTX) in patients with recurrent cerebral lymphoma. PATIENTS AND METHODS: Four patients with recurrent primary CNS lymphoma (PCNSL) were studied. Patients received 3.0 g/m2 MTX infused over 2 hours. Twelve hours after the start of MTX, 50 U/kg CPG2 was infused; a second dose of CPG2 was given 6 hours after the first. Blood and CSF were collected and assayed for levels of MTX, CPG2, and 2,4-diamino-N10-methylpteroic acid (DAMPA), a cleavage product of MTX after CPG2. Serum was collected for at least 2 weeks after administration of MTX-CPG2 to assess anti-CPG2 activity antibodies. RESULTS: All patients had at least a 2-log decline in plasma MTX levels to the subtherapeutic range within 5 minutes of CPG2 administration. The second dose of CPG2 did not further diminish the already low plasma MTX level. DAMPA appeared and was detected as the plasma MTX concentration decreased. CSF MTX concentration remained elevated for 4 hours after CPG2, and its decline followed first-order kinetics. Anti-CPG2 activity antibodies were not detected in any patient. No MTX or CPG2 toxicity was observed. CONCLUSION: CPG2 rescue is a safe, effective alternative to leucovorin rescue after HD MTX and may prove particularly useful for the treatment of MTX-sensitive CNS tumors, as it does not affect CSF MTX levels.

Successful carboxypeptidase G2 rescue in delayed methotrexate elimination due to renal failure.

We report on an 18.5-year-old woman with osteosarcoma and delayed methotrexate (MTX) elimination due to renal failure after high-dose MTX, in whom rescue with high doses of folinic acid caused intolerable side effects. In this life-threatening clinical situation, the patients was rescued by the administration of recombinant carboxypeptidase G2, a bacterial enzyme that rapidly hydrolyzes MTX into inactive metabolites. This is the first report on the successful clinical use of this alternative catabolic route for the elimination of MTX.

Effects of gamma-glutamyl hydrolase on folyl and antifolylpolyglutamates in cultured H35 hepatoma cells.

A subline of H35 hepatoma cells (H35D cells) that have been made resistant to 5,10-dideazatetrahydrofolate exhibits an increase in gamma-glutamyl hydrolase (GH) activity. GH is a lysosomal enzyme in H35 and H35D cells on the basis of comparison of the distribution of enzyme activity with other known lysosomal enzymes. The hydrolysis rate of methotrexate polyglutamate with isolated, intact lysosomes is 4-5-fold greater in H35D cells than in H35 cells. GH activity in isolated lysosomes is in part dependent on the presence of a reducing agent such as mercaptoethanol. Permeabilization of lysosomal preparations from both cell types by Triton X-100 causes a 10-fold enhancement in GH activity. The result of the enhanced activity of GH in H35D cells is a marked reduction in antifolylpolyglutamate concentration, with the parent antifolate being the predominant intracellular species found under all conditions tested. Unlike antifolates, the total intracellular folate concentration is nearly identical in both cells under standard culture conditions up to 10 microns folic acid. However, the chain length of folylpolyglutamates consists of predominantly triglutamates and tetraglutamates in H35D cells with increased GH, whereas it consists of pentaglutamates and hexaglutamates in the parental cells. At 50 and 100 microns folic acid, the folate accumulation in H35D cells is less than half that of H35 cells, and the predominant polyglutamate species in the H35D cells are the diglutamates through the tetraglutamates. The results demonstrate that the two H35 cell lines having equal folylpolyglutamate synthetase but that one with enhanced lysosomal GH activity exhibits a marked reduction in the amount and gamma-glutamyl chain length of folylpolyglutamates and antifolylpolyglutamates.

Carboxypeptidase-G2 rescue in a patient with high dose methotrexate-induced nephrotoxicity.

BACKGROUND: High dose methotrexate (HDMTX) induced renal failure is a medical emergency, as methotrexate (MTX) is primarily eliminated by renal excretion. High doses of leucovorin (LV) do not necessarily prevent toxicity in the presence of sustained elevated plasma MTX concentrations. The bacterial enzyme carboxypeptidase-G2 (CPDG2) hydrolyzes MTX into inactive metabolites and has been demonstrated to lower plasma MTX concentrations to nontoxic levels rapidly in the nonhuman primate after HDMTX infusion. Therefore, CPDG2 was evaluated as a rescue agent in a patient with acute renal dysfunction secondary to HDMTX: METHODS: A 16 year old patient with osteosarcoma experienced acute renal dysfunction after HDMTX administration, which resulted in markedly elevated and sustained plasma MTX concentrations. She received three doses of CPDG2 on the fifth day after HDMTX: Plasma MTX concentrations were determined before and after CPDG2 administration. RESULTS: The plasma MTX concentrations decreased from 60 to 1.2 microM within 15 minutes after the first dose of CPDG2. No rebound increase in plasma MTX concentrations or adverse reactions to the enzyme were observed. The patient developed only mild mucositis. Serum creatinine at the time of CPDG2 administration was 5 mg/dl and returned to normal within 7 weeks of enzyme administration. CONCLUSIONS: Carboxypeptidase-G2 rapidly, markedly, and persistently lowered plasma MTX concentrations to a level that could be rescued safely with LV. Based on the experience with this patient and on preclinical studies in nonhuman primates, CPDG2 appears to be more effective than hemodialysis or hemoperfusion, and may prove beneficial for patients at risk for life-threatening toxicity secondary to delayed excretion of MTX.

Antitumor effects of an antibody-carboxypeptidase G2 conjugate in combination with a benzoic acid mustard prodrug.

The F(ab')2 fragment of the antitumor monoclonal antibody, A5B7, was covalently linked to the bacterial enzyme carboxypeptidase G2 (CPG2). The resulting conjugate was used in combination with a prodrug of a benzoic acid mustard alkylating agent to treat human colon tumor xenografts in a two-step targeting strategy, antibody-directed enzyme prodrug therapy (ADEPT). The prodrug, 4-[(2-chloroethyl) (2-mesyloxyethyl)amino]-benzoyl-L-glutamic acid is rapidly converted by CPG2 to a drug that is at least 15x more toxic in vitro against LS174T colorectal tumor cells than the prodrug. Optimal tumor/blood ratios of the A5B7-CPG2 were achieved 72 h after administration of the conjugate to athymic mice bearing established LS174T tumor xenografts. Significant antitumor activity was seen in LS174T tumor-bearing mice treated with the conjugate followed 3 d later by the prodrug. In contrast, prodrug, conjugate, or active drug alone did not result in any antitumor activity in this tumor model. These studies demonstrate the advantage of a two-step ADEPT system for the treatment of colorectal cancer.

Methotrexate pharmacokinetics following administration of recombinant carboxypeptidase-G2 in rhesus monkeys.

PURPOSE: Carboxypeptidase-G2 (CPDG2) is a bacterial enzyme that rapidly hydrolyzes methotrexate (MTX) into inactive metabolites. As an alternative form of rescue after high-dose MTX (HDMTX), CPDG2 has more potential advantages than standard leucovorin (LV) rescue. In this study, the plasma pharmacokinetics of MTX with and without CPDG2 were evaluated in adult rhesus monkeys. MATERIALS AND METHODS: The plasma pharmacokinetics of MTX were determined in groups of animals that had received a 300-mg/m2 loading dose of MTX followed by a 60-mg/m2/h infusion during an 18-hour period. One group received CPDG2 at the end of the infusion, and the other group served as a control. Two additional animals with high titers of anti-CPDG2 antibody also were studied. RESULTS: During infusion, the steady-state MTX plasma concentration was 11.3 +/- 4.8 mumol/L. Without CPDG2, the postinfusion plasma MTX concentration remained above 0.1 mumol/L for more than 6 hours. After the administration of 50 U/kg of CPDG2, plasma MTX concentrations decreased to nontoxic levels (less than 0.05 mumol/L) within 30 minutes. The initial half-life (t1/2 alpha) of MTX decreased from 5.8 +/- 2.1 minutes to 0.7 +/- 0.02 minutes after enzyme administration. The postinfusion area under the plasma concentration time curve of MTX was 301 +/- 171 mumol/L/min without CPDG2 compared with 19.6 +/- 6.1 mumol/L/min with CPDG2. The immunogenicity studies performed indicated that although animals developed anti-CPDG2 antibodies, none of them manifested allergic symptoms. The effectiveness of CPDG2 was diminished but not eliminated in animals with high titers of anti-CPDG2 antibody. CONCLUSIONS: CPDG2 is capable of rapidly decreasing plasma MTX concentrations to nontoxic levels. The administration of CPDG2 seems safe, well tolerated, and it may be useful as an alternative to LV rescue.