Pharmacokinetics of glucarpidase in subjects with normal and impaired renal function.

Glucarpidase (formerly known as carboxypeptidase G2 or CPG2) is being evaluated for the adjunctive treatment of patients experiencing, or at risk of, methotrexate toxicity attributable to its delayed elimination. Delayed elimination of methotrexate can occur in patients with methotrexate-induced renal toxicity. In this study, glucarpidase pharmacokinetics were assessed in volunteer subjects with normal (n = 8) and severely impaired (n = 4) renal function. Each subject received a single intravenous dose of glucarpidase 50 U/kg (equivalent to 114.5 microg/kg) infused over 5 minutes. The mean maximum serum concentration (C(max)) for glucarpidase in renally impaired subjects was 2.9 microg/mL, the mean half-life (t(1/2)) was 10.0 hours, and the mean area under the serum concentration-time curve from time zero to infinity (AUC(0-infinity)) was 24.5 microg x h/mL. Similar values were found in subjects with normal renal function (mean C(max) 3.1 microg/mL, mean t(1/2) 9.0 hours, and mean AUC(0-infinity) 23.4 microg x h/mL). The results indicated little effect of renal impairment on the serum pharmacokinetics of glucarpidase.

Adecuación de las dosis de ácido fólico en la prevención de defectos congénitos / Folic acid dose in the prevention of congenital defects

Fundamento y objetivo: El ácido fólico (AF) sintético (usado en medicamentos y en la fortificación) está en forma de pteroilmonoglutamato (PGA), que no existe en la naturaleza, donde se encuentra como pteroilpoliglutamatos, especialmente en forma de 5-metiltetrahidrofolato. El organismo transforma el PGA en 5-metiltetrahidrofolato, y parece que este proceso de biotransformación del PGA se satura con dosis de 0,4 mg. Recientemente ha surgido la preocupación por el uso de dosis altas, ya que se superaría la capacidad metabólica del organismo, con la consiguiente acumulación plasmática de AF sintético no metabolizado. El objetivo de este estudio es analizar las dosis de AF sintético que se están utilizando en España y su evolución tanto temporal como espacial. Pacientes y método: Se ha analizado a 16.761 madres de recién nacidos sin defectos congénitos (controles) del Estudio Colaborativo Español de Malformaciones Congénitas (ECEMC). Se han considerado todas las formas de folatos sintéticos (fólico o folínico), que denominamos globalmente como AF. Resultados: Aunque se ha observado un significativo incremento secular de la proporción de madres que utilizaron AF en el último período (2003-2004), sólo un 17,37% tomó AF desde antes del embarazo. Del resto, un 71,13% lo tomó cuando ya estaban embarazadas y un 11,50% no lo tomó nunca. Además, de las madres que tomaron AF, más del 70% ingirió dosis de 4 mg o superiores. Los resultados por autonomías son muy similares a los globales, y en la mayoría de las comunidades más del 50% de las madres del último período tomaron dosis medias diarias entre 12,5 y 20 veces superiores a la recomendada de 0,4 mg/día. Conclusiones: La mayoría de las mujeres toman AF cuando ya están embarazadas o no lo toman nunca. La mayoría de las mujeres que toman AF ingieren dosis mucho más altas que las recomendadas. Los resultados de este trabajo muestran la necesidad de realizar una importante difusión de estos aspectos entre los profesionales sanitarios

Background and objective: Synthetic folic acid (FA) is in the form of pteroylmonoglutamate (PGA), a form that does not occur in nature, where it is in form of pteroylpolyglutamate, mainly as 5-methyltetrahydrofolate. The organism transforms PGA into 5-methyltetrahydrofolate, and it has been detected that this process is saturated at doses of 0.4 mg of PGA. Recently, some concern has been expressed on the use of higher than recommended doses of FA, because of the possibility of saturating the biotransformation process and the consequent plasmatic accumulation of unmetabolized synthetic FA. The objective of this study is to analyze the doses of synthetic FA that are being currently used in Spain, as well as its secular and geographic distribution. Patients and methods: The study was based on 16,761 mothers of non-malformed infants from the data base of the Spanish Collaborative Study of Congenital Malformations. All forms of folates (folic or folinic) have been considered under the general term of FA. Results: Although an increasing trend in the proportion of mothers using FA in the last study period (2003-2004), only 17.37% of the mothers had FA supplements since before pregnancy. Among the rest, 71.13% started using FA once they knew they were pregnant, and 11.50% never took this vitamin. In addition, more than 70% of mothers who took FA ingested doses of 4 mg or more. The results by Spanish Autonomic Regions are quite similar, with more that 50% of the mothers (of the period 2003-2004), in the great majority of the Regions, taking mean daily doses of FA that are between 12.5 and 20-fold higher than the recommended 0.4 mg/day. Conclusions: Most of women are having FA after conception, or do not ingest FA at all. Most of those women who take FA use doses much higher than recommended. The results of this study show that it is necessary to spread these aspects among the health professionals implicated

A phase I study of single administration of antibody-directed enzyme prodrug therapy with the recombinant anti-carcinoembryonic antigen antibody-enzyme fusion protein MFECP1 and a bis-iodo phenol mustard prodrug.

PURPOSE: Antibody-directed enzyme prodrug therapy is a two-stage treatment whereby a tumor-targeted antibody-enzyme complex localizes in tumor for selective conversion of prodrug. The purpose of this study was to establish optimal variables for single administration of MFECP1, a recombinant antibody-enzyme fusion protein of an anti-carcinoembryonic antigen single-chain Fv antibody and the bacterial enzyme carboxypeptidase G2 followed by a bis-iodo phenol mustard prodrug. MFECP1 is manufactured in mannosylated form to facilitate normal tissue elimination. EXPERIMENTAL DESIGN: Pharmacokinetic, biodistribution, and tumor localization studies were used to test the hypothesis that MFECP1 localizes in tumor and clears from normal tissue via the liver. Firstly, safety of MFECP1 and a blood concentration of MFECP1 that would avoid systemic prodrug activation were tested. Secondly, dose escalation of prodrug was done. Thirdly, the dose of MFECP1 and timing of prodrug administration were optimized. RESULTS: MFECP1 was safe and well tolerated, cleared rapidly via the liver, and was less immunogenic than previously used products. Eighty-fold dose escalation from the starting dose of prodrug was carried out before dose-limiting toxicity occurred. Confirmation of the presence of enzyme in tumor and DNA interstrand cross-links indicating prodrug activation were obtained for the optimal dose and time point. A total of 28 of 31 patients was evaluable for response, the best response being a 10% reduction of tumor diameter, and 11 of 28 patients had stable disease. CONCLUSIONS: Optimal conditions for effective therapy were established. A study testing repeat treatment is currently being undertaken.

Biodistribution of an antibody-enzyme conjugate for antibody-directed enzyme prodrug therapy in nude mice bearing a human colon adenocarcinoma xenograft.

The enzyme carboxypeptidase G2 (CPG2) can be targeted to tumors by antibodies and used to activate prodrugs in a treatment called antibody-directed enzyme prodrug therapy (ADEPT). Different doses of CPG2 conjugated to the anti-CEA antibody A5B7 were administered i.v. to nude mice bearing the LS174T human colon adenocarcinoma xenograft, and the biodistribution of conjugate activity 48 and 72 h later was determined using a novel high-performance liquid chromatography (HPLC) method. Conjugate doses of 2,500 and 625 U/kg gave tumor enzyme levels of 0.5-0.6 U/g. Lower doses of 300 and 150 U/kg gave tumor enzyme levels of 0.1-0.3 U/g. Intriguingly, the best tumor:blood ratio of conjugate activity at both 48 and 72 h was achieved after administration of the 625-U/kg dose, not the 2,500-U/kg dose. After 48 h this ratio was 3.8, whereas after 72 h the value was 5.5. This conjugate dose also gave the greatest tumor:tissue ratios in all other tissues examined. After 72 h the tumor:colon ratio was 105, whereas the tumor:kidney ratio was 36. In ADEPT, to obtain maximal tumor damage to LS174T xenografts in nude mice with minimal systemic toxicity using the A5B7-CPG2 conjugate, prodrug should therefore be administered at least 72 h after a conjugate dose of 625 U/kg.

Altered biodistribution of an antibody--enzyme conjugate modified with polyethylene glycol.

Polyethylene glycol modification of the antibody--enzyme conjugate, F(ab')2-A5B7-CPG2, extends its duration in the circulation of nude mice bearing human colonic cancer xenografts (LS174T). Increased concentration of modified conjugate is achieved in the tumour, but residual non-specific enzyme concentrations in normal tissue and blood demonstrate the fundamental requirement to remove or inactivate non-specifically held enzyme in this system.

Plasma clearance of an antibody--enzyme conjugate in ADEPT by monoclonal anti-enzyme: its effect on prodrug activation in vivo.

The effect of anti-enzyme antibody clearance on prodrug turnover in antibody-directed enzyme prodrug therapy (ADEPT) has been studied. Mice bearing LS174T xenografts were given localising carboxypeptidase G2 (CPG)2 conjugate (AEC) and 19 h later galactosylated anti-CPG2 antibody (SB43-GAL). In regimen I prodrug was injected 5 h after SB43-GAL as previously described. In regimen 2 and 3 a shortened and extended clearance time was used in which prodrug was administered 0.5 h or 53 h after SB43-GAL respectively. Regimen 1 resulted in similar tumour and normal tissue levels of active drug to those of the control in which prodrug was given 72 h after AEC. SB43-GAL therefore accelerated clearance of enzyme allowing early administration of prodrug. In regimen 2, very high active drug levels were found in the liver, showing removal of AEC from the blood followed by reactivation of enzyme and extensive and rapid prodrug turnover. Active drug levels in tumour and blood reached similar peak levels to those of the control. Regimen 3 resulted in lower active drug levels in tissues, consistent with degradation and excretion of enzyme. Regimen 3 also produced the best tumour to normal ratios for active drug. Residual prodrug in tumour was unaffected by SB43-GAL, showing the advantage of galactosylation in minimising inactivation of CPG2 in tumour. By contrast, residual prodrug in blood persisted for longer when SB43-GAL was used. Circulatory clearance of enzyme with SB43-GAL allows prodrug to be administered expediently with reduced toxicity and with the prospect of increasing the dosage.

In vitro and in vivo comparison of a randomly coupled antibody fragment-enzyme conjugate with a site-specific conjugate.

Two antibody fragment-enzyme conjugates, one obtained by random coupling of the two protein component, the other by site-specific ligation of the same component, were compared in vitro and in vivo for their usefulness in antibody directed enzyme prodrug therapy (ADEPT). The in vitro studies have shown that the site-specific conjugate has a higher antigen binding capacity, while both conjugates had similar specific enzymic activities. In vivo, the site-specific conjugate was cleared more rapidly. When correction was made for this faster clearance, both conjugates showed similar antitumor efficacy in a mouse xenograft system upon administration of a prodrug.

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.

Antibody directed enzyme prodrug therapy (ADEPT): a three phase system.

Monoclonal anti-CEA antibody, A5B7, and its fragments conjugated to CPG2 localize to a peak concentration in the LS174T xenografts within 24 h after injection, but enzyme activity persists in plasma such that prodrug injection has to be delayed for 5-6 days in order to avoid toxicity. Injection of prodrug at this time did not result in growth delay of this tumour. A three-phase system has been developed in which residual plasma enzyme was inactivated and cleared by a galactosylated anti-CPG2 antibody, SB43gal, allowing prodrug administration within 24 h after the conjugate. Using this three-phase system, a marked growth delay of this tumour was achieved after a single course of treatment consisting of conjugate injection followed by SB43gal, 19 h later and three doses of the prodrug.

Inactivation and clearance of an anti-CEA carboxypeptidase G2 conjugate in blood after localisation in a xenograft model.

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.

The potential of carboxypeptidase G2: antibody conjugates as anti-tumour agents. II. In vivo localising and clearance properties in a choriocarcinoma model.

The in vivo localising and clearance properties of conjugates of the folate-degrading enzyme carboxypeptidase G2 (CPG2) with anti-human chorionic gonadotrophin (W14A) were measured in nude mice bearing CC3 choriocarcinoma xenografts. Conjugates of W14A-F (ab')2 fragment coupled to CPG2 localised in tumour as effectively as native antibody alone but showed lower uptake in other major tissues. The clearance rates of conjugates prepared with intact antibody or F (ab')2 fragment were shown to be up to five-fold faster than for native antibody and two-fold compared to F (ab')2 fragment. Molecular weight analysis of residual conjugate in the blood showed that no degradation of conjugate to its component molecules occurred during circulation. It was concluded that F (ab')2: CPG2 conjugates offered the greatest potential for targeting applications.