J Clin Pharmacol
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PHARMACOKINETICS/SPECIAL POPULATIONS

Pharmacokinetics of Glucarpidase in Subjects With Normal and Impaired Renal Function

Marc Phillips, PhD, William Smith, MD, FACC, Guhan Balan, PhD and Suzanne Ward, PharmD

From Protherics Medicines Development Ltd, The Heath Business & Technical Park, Runcorn, Cheshire, UK (Dr Phillips); New Orleans Center for Clinical Research, New Orleans, LA (Dr Smith); Procter & Gamble Pharmaceuticals, Mason, OH (Dr Balan); and Protherics Inc, Brentwood, TN (Dr Ward).

Address for correspondence: Marc Phillips, PhD, Protherics PLC, The Heath Business & Technical Park, Runcorn, Cheshire, WA7 4QX, UK.


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
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 µg/kg) infused over 5 minutes. The mean maximum serum concentration (Cmax) for glucarpidase in renally impaired subjects was 2.9 µg/mL, the mean half-life (t1/2) was 10.0 hours, and the mean area under the serum concentration-time curve from time zero to infinity (AUC0-{infty}) was 24.5 µgxh/mL. Similar values were found in subjects with normal renal function (mean Cmax 3.1 µg/mL, mean t1/2 9.0 hours, and mean AUC0-{infty} 23.4 µgxh/mL). The results indicated little effect of renal impairment on the serum pharmacokinetics of glucarpidase.

Key Words: Glucarpidase • methotrexate • renal • Voraxaze

Methotrexate (MTX, amethopterin, 4-amino-N10-methyl pteroylglutamic acid) is a cytotoxic agent that exerts its effect by competitively inhibiting dihydrofolate reductase, the intracellular enzyme responsible for converting folic acid to reduced folate cofactors, which are necessary for DNA synthesis. MTX has been in clinical use since 19481 and is used alone or as part of a combined chemotherapy regimen, in either standard or high doses, in the treatment of a range of cancers. It is also used at lower doses to treat other diseases.2,3 MTX is almost exclusively cleared via the kidneys,4 and administration of high-dose MTX (HDMTX, defined as doses ≥1 g/m2) results in high concentrations of unchanged MTX in urine, which may lead to various degrees of renal damage in some patients. This damage delays the elimination of MTX, thus extending the duration and magnitude of systemic exposure to MTX. Serious toxicity following HDMTX therapy (such as myelosuppression and severe mucositis) is related to the duration and magnitude of systemic exposure to MTX.5 Delayed MTX elimination produces toxicity, because it results in persistently elevated systemic MTX concentrations.6,7 In early studies with HDMTX, severe toxicity occurred in approximately 10% of patients, and there was a 6% mortality rate.8

HDMTX usually can be administered safely to patients with normal renal function by using concomitant vigorous hydration and alkalinization of the urine, routinely monitoring serum MTX, and using leucovorin (LV) calcium (a technique commonly known as leucovorin or folinate rescue) to prevent potentially lethal MTX toxicity. However, MTX-induced nephrotoxicity still occurs in a fraction of patients, especially in adult patients with poor performance status.2,9 If untreated, the acute effect on renal function can lead to delayed clearance of MTX, ineffective rescue by LV, and a host of secondary toxicities.10

Glucarpidase (formerly known as carboxypeptidase G2 or CPG2) is a bacterial enzyme that cleaves the MTX molecule into inactive metabolites, 4-deoxy-4-amino-N10-methylpteroic acid (DAMPA) and glutamate, which are metabolized by the liver. Use of glucarpidase thus provides an alternative route of MTX elimination, which is particularly important in patients who develop renal dysfunction attributable to MTX nephrotoxicity.11-14

The effect of glucarpidase on the kinetics of MTX elimination in cancer patients has been reported elsewhere.15-18 It has not been feasible to investigate the kinetics of glucarpidase in patients, because those who receive it are in a potentially life-threatening situation, and taking serial blood samples at predefined time points is not practical. Therefore, the kinetics of glucarpidase has been investigated in healthy subjects, in the absence of MTX. The present study examined the pharmacokinetics of glucarpidase in subjects who had both normal and severely impaired renal function, the latter to more closely match the intended patient population.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Subjects
Twelve subjects aged between 22 and 51 years participated in this study; 8 subjects (median age 42 years) had normal renal function (calculated creatinine clearance >80 mL/min) and 4 subjects (median age 41 years) had severe renal impairment (calculated creatinine clearance <30 mL/min). Subjects with normal renal function had a median weight of 83.4 kg (range 67.4-114.7 kg), and subjects with impaired renal function had a median weight of 87.5 kg (range 75.3-106.5 kg). Nine subjects (6 with normal renal function and 3 with impaired renal function) were male. Three subjects (2 with normal renal function and 1 with impaired renal function) were female. The self-reported races of the 12 subjects were 9 Black (6 with normal renal function and 3 with impaired renal function) and 3 Caucasian (2 with normal renal function and 1 with impaired renal function). Demographic details are shown in Table I. All subjects gave their written informed consent prior to commencing the study. The study protocol and consent form were approved by the Crescent City Institutional Review Board (New Orleans, Louisiana).


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  		Table I Subject Demographics

 

Design
This was an open-label, single-site study to characterize the pharmacokinetics and assess the safety and tolerability of glucarpidase. A study of this size was considered adequate to characterize the pharmacokinetics of glucarpidase and to determine whether the pharmacokinetics are affected by renal impairment.

Test Material
A single intravenous dose of glucarpidase (50 U/kg, VoraxazeTM lot number 2090302, Cangene Corporation, Winnipeg, Canada) was administered to each study subject. Prior to administration, each vial of glucarpidase was reconstituted with 1.0 mL of sterile normal saline solution (sodium chloride 0.9% wt/vol). Following reconstitution, each milliliter contained approximately 1000 U of glucarpidase. Glucarpidase was reconstituted immediately prior to use and administered via bolus intravenous injection over 5 minutes.

Sample Collection for Pharmacokinetic Analyses
Serum glucarpidase profiles were evaluated for all subjects using 7-mL blood samples collected pre-dose (immediately prior to the administration of glucarpidase), at the end of the 5-minute infusion of glucarpidase, and 0.25, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 48, 72, and 96 hours following the start of the glucarpidase infusion. Urinary excretion of glucarpidase was evaluated using samples obtained in the following intervals: 0 to 2, 2 to 4, 4 to 8, and 8 to 24 hours following the start of glucarpidase infusion.

Glucarpidase Concentration Measurements
Serum concentrations of glucarpidase were determined by Covance Laboratories Ltd (Harrogate, UK) by assaying both enzymatic activity using ultraviolet spectroscopy and total glucarpidase protein using a competitive enzyme-linked immunosorbent assay (ELISA). Urinary excretion of unchanged glucarpidase was evaluated using the ELISA method only.

The ELISA was a competitive enzyme immunoassay in which goat anti-rabbit antibodies are used to capture a specific glucarpidase complex in each sample. A biotinylated conjugate and glucarpidase in sample or standards compete for these specific binding sites. Because the concentration of glucarpidase increases, the amount of biotinylated conjugate captured by the antibody decreases. This amount is detected using a streptavidin alkaline phosphatase conjugate and a chromatogenic substrate reaction and is measured at 492 nm. The amount of glucarpidase in each sample is determined by comparing results from the sample wells with a standard curve. The lower limit of quantitation (LLOQ) of this assay was 0.075 µg/mL for serum and 0.012 µg/mL for urine. The interassay precision of the assay, as determined from quality control samples, was 28% or better.

In the enzymatic activity assay, the activity of glucarpidase in human serum is determined using an enzyme-substrate photometric method, in which the cleavage of methotrexate is monitored by ultraviolet absorbance at 320 nm against time. The LLOQ of this assay was 0.26 µg/mL, and the interassay precision of quality control samples was 15.2% or better.

Pharmacokinetic Analyses
The following pharmacokinetic (PK) parameters were calculated, for both serum enzyme activity and serum ELISA data, according to the model independent approach19: maximum serum concentration (Cmax), time to maximum concentration (Tmax), area under the serum concentration-time curve from time zero to last measurable time point (AUC0-t), area under the serum concentration-time curve from time zero to infinity (AUC0-{infty}), magnitude of the slope of the linear regression of the log concentration versus time profile during the terminal phase ({lambda}z), half-life (t1/2), total clearance (CL), and steady-state volume of distribution (Vss), PK parameters were not calculated for urine samples because only 1 subject had measurable concentrations.

Analysis of variance (ANOVA) was performed on natural log (ln) transformed Cmax, AUC0-t, AUC0-{infty}, and t1/2 to compare normal and renally impaired subjects. This analysis was performed separately for both the enzymatic and the ELISA methods. For all PK parameter calculations, serum concentrations that were below the lower limit of quantification were treated as zero values if they were present at the beginning or the end of the profile. In all other cases, LLOQ values were treated as missing values. All subjects were dosed and infused correctly; hence, a dose of 50 U/kg glucarpidase (equivalent to 114.5 µg/kg) and a 5-minute infusion time were used in all serum PK calculations. Actual sampling times were used in the calculation of serum PK parameters. All PK calculations were performed using WinNonlin® (Pharsight Corporation, Mountain View, California, version 4.1) and/or SAS® (version 9.2; SAS Institute, Cary, North Carolina).

Safety Assessment
The safety and tolerability of glucarpidase were monitored by collection of adverse event reports, physical examinations, vital signs, electrocardiograms, and clinical laboratory evaluations (hematology, clinical chemistry, and urinalysis).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
All 12 enrolled subjects completed the study. The PK data for serum glucarpidase from both subject groups using both assay methods are shown in Table II. The mean concentration (±SEM) time course data from both groups using the ELISA method are shown in Figure 1.


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  		Table II Serum Pharmacokinetics for Glucarpidase

 

Figure 1
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  		Figure 1. Mean concentration-time profile of serum glucarpidase using the enzyme-linked immunosorbent assay. Mean glucarpidase serum concentration (±SEM, as indicated by the error bars) up to and including the 48-hour sample point. Serum samples were also drawn at 72 and 96 hours; in each case the serum glucarpidase concentration was <LLOQ and is thus not plotted.

 


Figure 2
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  		Figure 2. Association between creatinine and glucarpidase clearance.

 
A comparison of the pharmacokinetic parameters showed no evidence of a difference between the 2 subject groups (P > .1, ANOVA; see Table III). The lack of association between creatinine clearance and total glucarpidase clearance is plotted in Figure 2. For consistency, the creatinine clearance was normalized by body weight prior to plotting.


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  		Table III Summary of the Comparison of Glucarpidase Pharmacokinetics Between Subjects With Normal and Impaired Renal Function

 

Eleven of the 12 subjects had no quantifiable excretion of unchanged glucarpidase in urine. In 1 (renally impaired) subject, the concentrations of unchanged glucarpidase measured in the urine between 0 and 2 hours and between 2 and 4 hours were 13.3 and 15 ng/mL, respectively, which equates to 0.000568% when expressed as a fraction of the dose infused.

No adverse events were reported during this study. No changes in hematology, chemistry, urinalysis, or vital signs were observed postdose, and no significant differences were observed between subjects with normal renal function and subjects with renal impairment. There were no abnormal, clinically significant electrocardiographic findings for any subject.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
This is the first study explicitly designed to elucidate the pharmacokinetics of glucarpidase in human subjects. However, glucarpidase pharmacokinetics were also assessed in a small pilot study in patients with central nervous system lymphoma,17 in which glucarpidase concentrations were assessed using an enzyme activity assay. These patients were treated with HDMTX, and it is possible that the presence of MTX and its metabolites may have affected the assay measurements. Nevertheless, the results found by De Angelis et al17 appear to be comparable to those of this study.

Because the ELISA method was specific to glucarpidase, PK parameter results using the ELISA method were considered as primary in summarizing the findings of this study. These results indicated about a 7% lower mean Cmax for glucarpidase in subjects with impaired renal function relative to the normal renal function group; the total glucarpidase exposure, as measured by mean AUC0-{infty}, was marginally higher by about 5% in subjects with impaired renal function. However, no significant differences were noted in mean Cmax and AUC0-{infty} between the 2 subject groups.

Median Tmax was short for both subject cohorts, indicating rapid equilibration of glucarpidase after completion of the short infusion. The ELISA results revealed no significant differences in mean t1/2 between the 2 subject groups. Large variability was noted, especially for AUC0-t and AUC0-{infty}, in the renally impaired subject group, with 1 subject having a higher exposure relative to the other 3 subjects. This finding may, however, be attributable to the limited number of subjects in this group (n = 4).

Our results indicate that glucarpidase at a dose of 50 U/kg is well tolerated in subjects with both normal and impaired renal function. Furthermore, the serum pharmacokinetics of glucarpidase are unaltered in subjects with renal impairment and therefore are not expected to be altered in the target population.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
Financial disclosure: None declared.

DOI: 10.1177/0091270007311571


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

1. Bleyer WA. The clinical pharmacology of methotrexate. Cancer. 1978;41: 36-51.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

2. Ackland SP, Schilsky RL. High-dose methotrexate: a critical reappraisal. J Clin Oncol. 1987;5: 2017-2031.[Abstract/Free Full Text]

3. Frei E III, Blum RH, Pitman SW, et al. High dose methotrexate with leucovorin rescue: rationale and spectrum of antitumor activity. Am J Med. 1980;68: 370-376.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

4. Bleyer WA. Methotrexate: clinical pharmacology, current status and therapeutic guidelines. Cancer Treat Rev. 1977;4: 87-101.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

5. Von Hoff DD, Penta JS, Helman LJ, Slavik M. Incidence of drugrelated deaths secondary to high-dose methotrexate and citrovorum factor administration. Cancer Treat Rep. 1977;61: 745-748.[Web of Science][Medline] [Order article via Infotrieve]

6. Abelson HT, Fosburg MT, Beardsley GP, et al. Methotrexate-induced renal impairment: clinical studies and rescue from systemic toxicity with high-dose leucovorin and thymidine. J Clin Oncol. 1983;1: 208-216.[Abstract]

7. Condit PT, Chanes RE, Joel W. Renal toxicity of methotrexate. Cancer. 1969;23: 126-131.[CrossRef][Medline] [Order article via Infotrieve]

8. Stoller RG, Hande KR, Jacobs SA, Rosenberg SA, Chabner BA. Use of plasma pharmacokinetics to predict and prevent methotrexate toxicity. N Engl J Med. 1977;297: 630-634.[Abstract]

9. Widemann BC, Balis FM, Murphy RF, et al. Carboxypeptidase-G2, thymidine, and leucovorin rescue in cancer patients with methotrexate-induced renal dysfunction. J Clin Oncol. 1997;15: 2125-2134.[Abstract/Free Full Text]

10. Messmann RA, Allegra CJ. Antifolates. In: Cancer Chemotherapy and Biotherapy: Principles and Practice. Chabner B, Longo DL, eds. Baltimore, MD: Lippincott Williams & Wilkins; 2001: 139-184.

11. Adamson PC, Balis FM, McCully CL, et al. Rescue of experimental intrathecal methotrexate overdose with carboxypeptidase-G2. J Clin Oncol. 1991;9: 670-674.[Abstract]

12. Mohty M, Peyriere H, Guinet C, et al. Carboxypeptidase G2 rescue in delayed methotrexate elimination in renal failure. Leuk Lymphoma. 2000;37: 441-443.[Web of Science][Medline] [Order article via Infotrieve]

13. Von Poblozki A, Dempke W, Schmoll HJ. Carboxypeptidase-G2-rescue in a woman with methotrexate-induced renal failure. Med Klin. 2000;95: 457-460.[CrossRef][Web of Science]

14. Widemann BC, Sung E, Anderson L, et al. Pharmacokinetics and metabolism of the methotrexate metabolite 2, 4-diamino-N(10)-methylpteroic acid. J Pharmacol Exp Ther. 2000;294: 894-901.[Abstract/Free Full Text]

15. Adamson PC, Balis FM, Boron M, et al. Carboxypeptidase-G2 (CPDG2) and leucovorin (LV) rescue with and without addition of thymidine (Thd) for high-dose methotrexate (HDMTX) induced renal dysfunction. J Clin Oncol. 2005;23(16 suppl): 2076.

16. Buchen S, Ngampolo D, Melton RG, et al. Carboxypeptidase G2 rescue in patients with methotrexate intoxication and renal failure. Br J Cancer. 2005;92: 480-487.[Web of Science][Medline] [Order article via Infotrieve]

17. De Angelis LM, Tong WP, Lin S, Fleisher M, Bertino JR. Carboxypeptidase G2 rescue after high-dose methotrexate. J Clin Oncol. 1996;14: 2145-2149.[Abstract/Free Full Text]

18. Schwartz S, Borner K, Muller K, et al. Rescue with recombinant carboxypeptidase G2 (CPG2) in high-dose methotrexate (HD-MTX) induced renal failure: results in 42 patients from a German emergency protocol. J Clin Oncol. 2004;22(14 suppl): 745-c.[Free Full Text]

19. Gibaldi M, Perrier D. Pharmacokinetics. 2nd ed. New York, NY: Marcel Dekker; 1982.
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This Article
Right arrow Abstract Freely available
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0091270007311571v1
48/3/279    most recent
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