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Keywords:

  • asparaginase;
  • asparagine;
  • acute lymphatic;
  • leukaemia;
  • childhood;
  • clinical pharmacology

For over 40 years, the enzyme asparaginase has held an established position in therapy concepts employed in the treatment of lymphoproliferative disease of childhood.

Owing to the fact that asparaginase is the only therapeutically applied enzyme preparation and is not associated with any remarkable haematotoxicity, it is considered an appropriate partner in combination chemotherapy regimens of many highly active treatment protocols. Nevertheless, or quite possibly for this very reason, there continues to be considerable uncertainty as to the best application scheme. This uncertainty covers relevant aspects of clinical practice, such as dosage, dose intervals, optimal positioning within the treatment schedule, optimal duration of one treatment course, or interchangeability of and appropriate indications for different asparaginase preparations.

The expectations implied in the title of this paper must thus be qualified, in that current knowledge supplies no general standard for the ‘best way’ of using asparaginase. It is, however, possible to weigh the options in a rational manner and approach the definition of a ‘proper way of use’ via a largely consistent construct, which adds a high degree of plausibility to the data available on asparaginase. Surrogate parameters of the pharmacokinetic (PK) and pharmacodynamic (PD) behaviour of asparaginase play an important role, as they represent the interface between preclinical and clinical findings, and reflect the treatment intensity achievable with asparaginase.

The present review is thus intended to approach the ‘best way of use’, starting with the clinico-pharmacological aspects. First, the conceptual frame will be described, which enables us to develop definitions for a rational use of asparaginase. The second part will deal with the asparaginase preparations used in clinical practice – with a focus on the pharmacological surrogates – and will attempt to draw relevant conclusions from available findings.

Mechanism of action

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

Asparaginase catalyses the hydrolytic decomposition of the amino acid asparagine to aspartic acid and ammonia. When applied for medication, the enzyme leads to a reduction in the asparagine level and, when appropriately scheduled, to persistent depletion of this amino acid which is non-essential under physiological conditions.

According to the conceptions on the mechanism of action proposed around the middle of the 20th century, and still valid today, the anti-leukaemic effect of asparaginase is associated with a particular characteristic of the malignant lymphoblastic cells’ metabolic deficiency.

Malignant blasts, in contrast to most of the healthy tissues, show a reduced expression of the enzyme asparagine synthetase and are thus unable to synthesize sufficient amounts of asparagine. As a consequence, the blasts depend on extracellular sources of asparagine to maintain protein biosynthesis (Haley et al, 1961; Prager & Bachynsky, 1968; reviews: Cooney & Handschumacher, 1970; Wriston & Yellin, 1973). An asparaginase-induced interruption of the asparagine supply leads to a depletion of the substrate needed by the malignant blasts. The resulting impairment of protein biosynthesis and subsequent cell cycle arrest finally lead to cell death through cellular dysfunction (Becker & Broome, 1967, 1969).

Alternative approaches to explain the mechanism of action primarily discuss the induction of apoptosis by way of signal transduction (Story et al, 1993; Ueno et al, 1997). As those ideas have not yet been developed into a differentiated concept and still lack relevance for clinical application, they will not be considered in this review.

So far, there is neither in vivo nor in vitro knowledge about the duration of asparagine depletion necessary for blasts to undergo irreversible changes. The time of exposure that is needed for killing cells strongly depends on the protocol-specific context of the drug. As the different study protocols have their own empirically based, complex histories of development, it is hardly possible to state anything about a cumulative single drug exposure.

PK profile of asparaginase preparations available for clinical application

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

The preparations approved for clinical application are based on asparaginases derived from bacterial sources of either Escherichia coli or Erwinia chrysanthemi (Mashburn & Wriston, 1964; Wade et al, 1968). The enzyme is available either chemically unchanged (native asparaginases) or conjugated to polyethylene glycol (PEG asparaginase). Many older publications even fail to name the particular asparaginase preparation used. On the contrary, E. coli asparaginase preparations from different sources do in fact differ in their physicochemical and PK properties (e.g. review: Wriston & Yellin, 1973). As a consequence, it is fairly difficult to devise general recommendations based on those older reports.

In clinical practice, the enzyme is currently given by the intravenous (i.v.; e.g. Schrappe et al, 2000) or intramuscular (i.m.) route (e.g. Gaynon et al, 2000), or subcutaneously (e.g. Eden et al, 2000). Although this might theoretically result in different PK profiles, information on the comparability of the routes of administration is limited with regard to the currently available preparations.

All asparaginases, due to their high molecular weight, are distributed intravascularly and are generally unable to cross the blood–brain barrier (Ohnuma et al, 1970). Only after the application of very high doses of an E. coli asparaginase could low levels of enzyme activity be detected in the cerebrospinal fluid (CSF) (Schwartz et al, 1970; Riccardi et al, 1981). The ratio between the enzyme activity measured in the CSF and the activity in the blood was found to be <0·2% (Riccardi et al, 1981).

Conclusive findings on the metabolism and elimination of asparaginase have not yet been produced. Various studies indicate that it might be eliminated via the reticulo-endothelial system (Ohnuma et al, 1970; Brueck et al, 1989). PK models describe first order elimination (Schwartz et al, 1970; Asselin et al, 1993; Keating et al, 1993; Albertsen et al, 2001a; Avramis et al, 2002). As regards PEG asparaginase, elimination following Michaelis–Menten kinetics is alternatively favoured (Müller et al, 2000). All studies, however, unvaryingly show high intra-individual variability of the kinetics, which is why prospective statements on the course of activity in individual patients are hardly possible. Apart from the classical PK variables, such as bodyweight, height and sometimes co-medication, the suppression of the reticulo-endothelial system and the presence of asparaginase antibodies will most probably exert some influence on the elimination process. Generally, the immune reaction and the formation of specific antibodies are essential factors in the discussion on the use of asparaginase (e.g. Woo et al, 1998). The probability of those reactions occurring is considered to be related to the number of doses within one treatment phase and, above all, to repeat application following an extended asparaginase-free interval (review: Müller & Boos, 1998). Clinically, the formation of antibodies may manifest itself by hypersensitivity reactions (Killander et al, 1976; Evans et al, 1982; Müller et al, 2001) or may lead to a faster decrease of enzymatic activity, an effect which is of relevance from a pharmacological point of view (Kurtzberg et al, 1993; Kurtzberg, 1994; Asselin, 1999). The latter phenomenon has been appropriately termed ‘silent inactivation’ for its lack of clinical symptoms. While the described reactions of the immune system may develop independently of each other, each one necessitates the discontinuation of the particular asparaginase used. Owing to comparatively low immunological cross reactivity between the available Erwinia chrysanthemi- and E. coli-based preparations, treatment may be continued after switching to an asparaginase preparation from a different bacterial source (Ohnuma et al, 1972; Billett et al, 1992), or to a pegylated preparation when the immune reaction occurred with a native E. coli preparation (review: Holle, 1997). The cross reactivity of the different native E. coli sources has never been investigated. The first results of current in vitro studies suggest cross reactivity between antibodies against native and pegylated E. coli preparations and no cross reactivity with regard to Erwinia asparaginase (Wang et al, 2003).

Relationship between PK and PD

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

Previous studies focussing on the interplay between the PK and PD of asparaginase have probably supplied the most relevant stimuli for the eventual definition of a ‘best way of use’. Findings from a primarily animal experimental study provided the basis for postulating that the peripheral application of asparaginase will lead to asparaginase activities in the serum of >100 U/l and the subsequent complete depletion of asparagine from both plasma and CSF (Riccardi et al, 1981). At present, there are no publications in the literature giving clear information on the mechanisms that induce the depletion of asparagine from CSF under asparaginase treatment. The balancing of amino acid concentrations by diffusion into the systemic circulation due to the different gradients of CSF and plasma has been proposed as one explanation (Schwartz et al, 1970; Müller & Boos, 1998). Minute levels of asparaginase in the CSF below the detection level might also be responsible for the depletion of the physiologically low levels of CSF asparagine (around 3–8 μmol/l) (Gerrits et al, 1989; Woo et al, 1999), especially, as they are not exposed to the sketched elimination processes in this particular compartment.

Faced with complex treatment protocols that regularly employ combination chemotherapy, findings on the surrogates of asparaginase activity and asparagine concentration have gained relevance in the assessment of asparaginase treatment. While those parameters have not yet been validated for efficacy, they are still considered to be useful tools in estimating the treatment intensity achieved. Using those surrogates is supported by considerations of practicability. The target ranges to be achieved by asparaginase treatment can be defined for each parameter. Treatment objectives may be considered to be met when the asparagine concentration in the blood or CSF is no longer detectable or when the asparagine activity in the blood exceeds 100 U/l. Moreover, a range of analytical methods is available that cover the relevant ranges for all parameters. The lower limit of quantification is c. 0·2 μmol/l for the asparagine concentration (e.g. Boos et al, 1996) and c. 2·5 U/l for asparaginase activity (Lanvers et al, 2002). With a reference range of 40–80 μmol/l for plasma asparagine, depletion by more than two log steps can thus be detected (Lepage et al, 1997). However, the minimum level not to be exceeded for a ‘therapeutic’ effect has not been determined yet. Considering that low physiological CSF levels of 3–8 μmol/l suffice for blast growth, aiming at complete asparagine depletion on the basis of the above limits of quantification appears reasonable.

Summary

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References
  • Asparaginase treatment aims at the complete depletion of the amino acid asparagine.
  • Persistent asparagine depletion can be achieved with serum asparaginase activities >100 U/l.
  • Analytical methods are available for measuring the surrogates of asparaginase activity and asparagine concentration.
  • The time course of asparaginase activity may be influenced by antibody formation in a variety of ways, and there may be ‘silent inactivation’ without clinical symptoms.
  • ‘Silent inactivation’ results in reduced treatment intensity, a risk factor potentially jeopardizing the desired treatment result.
  • In the event of a hypersensitivity reaction, asparaginase treatment may be continued after switching preparations.

Single drug application

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

Asparaginases of medicinal efficacy, i.e. the native enzymes isolated from E. coli and Erwinia chrysanthemi, have been available since the mid-1960s. Considerations as to the ‘proper way of use’ of asparaginase, however, were initially postponed as the first clinical trials focused on proof of efficacy. In fact, single drug application of (mostly E. coli-derived) asparaginase was found to result in response rates of up to 65% with remissions lasting around 60 d (Oettgen et al, 1967; Ohnuma et al, 1970; Tallal et al, 1970; Jaffe et al, 1971; Sutow et al, 1971). For the most part, those studies were carried out in paediatric patients with acute lymphatic leukaemia (ALL) relapse and employed the E. coli-derived enzyme in different treatment schemes.

Dose finding studies

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

The first clinical studies, which focused on a systematic approach towards the best scheme of asparaginase application and dosage, were run within the framework of three consecutive treatment protocols of the Children's Cancer Study Group (CCSG) between 1968 and 1975 (Ertel et al, 1979; Nesbit et al, 1979, 1981). The patient group included children with ALL who had previously received chemotherapy for their disease and had relapsed. One randomized trial of various schemes of application, differing with regard to dose and i.m. versus i.v. mode of application, tested the E. coli preparation CrasnitinTM (Bayer, Leverkusen, Germany; no longer marketed). The main conclusion from the results of this trial was that a schedule providing i.m. doses of 6000 IU/m2 body surface area CrasnitinTM three times/week over 4 weeks could be regarded as the most effective scheme. With this schedule remission rates of c. 60% were achieved, a rate that could not be further improved by higher doses.

The findings of the CCSG studies on CrasnitinTM subsequently entered all further considerations regarding the definition of the ‘best way of use’. In the process, the asparaginases from E. coli and Erwinia chrysanthemi were usually applied according to identical schemes, although (i) comparable results from dose finding studies were unavailable for the Erwinia chrysanthemi asparaginase, (ii) different isolated asparaginases had already been found to exhibit different physico-chemical properties (Ohnuma et al, 1970; review: Wriston & Yellin, 1973), and (iii) the first data on significant differences in the PK profile of various preparations had already been published (Schwartz et al, 1970). The choice of the particular asparaginase preparation used for front-line therapy in a given treatment protocol was primarily governed by aspects such as regional availability and individual clinical experience.

Pharmacological surrogates and drug monitoring

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

New stimuli in the discussion of a ‘proper way of use’ of the native preparations stemmed from studies using the surrogate parameters of asparaginase activity and asparagine concentration from the early 1990s onward.

Treatment approaches that applied different asparaginases in children with newly diagnosed ALL were the first to reveal significant differences of their PK properties. Whereas the E. coli preparation ElsparTM (Merck, West Point, PA, USA) was found to result in a half-life of enzyme activity of 1·24 ± 0·17 d in the blood, the half-life of the identically applied Erwinia chrysanthemi asparaginase ErwinaseTM (Ipsen, Maidenhead, UK) was 0·65 ± 0·13 d (mean ± standard deviation in both cases). Similar differences were also found with respect to the duration of asparagine depletion (14–23 d with ElsparTM vs. 7–15 d with ErwinaseTM) (Asselin et al, 1993; Asselin, 1999).

Data on the time course of the asparagine concentration in the CSF have been reported for the E. coli preparation ElsparTM in paediatric ALL patients (Woo et al, 1999). Repeated doses of i.m. 10 000 IU/m2 led to median asparagine concentrations below the detection limit during treatment and up to 5 d after the last dose. The percentage of patients with CSF levels below the detection limit under treatment was c. 74% (17 of 23 patients).

Important findings on the ‘proper way of use’ of asparaginase resulted from a drug monitoring programme established within the framework of the paediatric ALL protocols of the Berlin–Frankfurt–Münster (BFM) group. Conclusions on the course over time of asparaginase activity and asparagine concentration were drawn for three drug products available at the time (CrasnitinTM and MedacTM (Medac, Hamburg, Germany; produced by Kyowa Hakko, Tokyo, Japan)) from E. coli, ErwinaseTM from Erwinia chrysanthemi), and different dosage schemes and dosage recommendations were modified accordingly (Boos et al, 1996).

One main finding from those investigations concerned the significant difference between the three preparations regarding enzyme activity and the effect on the plasma asparagine concentration. The established dosage scheme, which was identical for all three asparaginases, recommended the i.v. application of 10 000 IU/m2 at, usually, 3-d intervals. It was found that both of the E. coli products, CrasnitinTM and MedacTM, when given for induction, led to complete depletion of plasma asparagine as measured prior to the next dose; MedacTM, however, showed a markedly higher median enzyme activity in the serum than CrasnitinTM (475 U/l vs. 74 U/l). Similar observations were made during reinduction, where median enzyme activities were 528 U/l for MedacTM and 49 U/l for CrasnitinTM on day 3 after the application. In those cases where the medication was switched to ErwinaseTM as a result of hypersensitivity reactions, the median activity was below the analytical limit of quantification of 20 U/l. The asparagine concentration in the plasma showed a corresponding behaviour: complete depletion was detected in more than 90% of the MedacTM and over 60% of the CrasnitinTM samples, as opposed to only 26% of the ErwinaseTM samples.

Similar results with ErwinaseTM were obtained in studies on asparagine depletion within the framework of an Italian ALL protocol, where the product was used as first-line therapy (Gentili et al, 1996). Using a dosage scheme identical with the one employed by the ALL-BFM study, this study reported complete depletion of plasma asparagine during reinduction in 25% of cases. About 80% of patients in this study showed complete depletion during the induction phase. Another study associated with the Italian ALL protocol, which investigated the i.v. versus i.m. application of both MedacTM and ErwinaseTM, revealed no difference between the two modes of application (Rizzari et al, 2000). Over 90% of the MedacTM and c. 40% of the ErwinaseTM group showed enzyme activities in the serum above the study-specific threshold of 100 U/l prior to the next dose. Both of the drug products achieved complete depletion of asparagine from plasma and CSF in all samples measured.

The drug monitoring programme described above (Boos et al, 1996) prompted further studies on the dosage scheme of MedacTM and ErwinaseTM in association with the ALL-BFM protocols. Those investigations were aimed at achieving a treatment intensity that was comparable with the one reached by the traditionally employed CrasnitinTM. The studies on MedacTM, which revealed high inter- and intra-individual variability of serum asparaginase trough activities (range 83–552 U/l), led to the recommendation of reducing the dose to 5000 IU/m2; this recommendation took into consideration that the complete depletion of asparagine from blood and CSF samples had already been observed after a dose of 2500 IU/m2 in 15/15 and 18/18 patients respectively (Ahlke et al, 1997). With ErwinaseTM, the intended treatment intensity was achieved with a dose of 20 000 IU/m2 and shorter, i.e. 2-d dose intervals (Vieira Pinheiro et al, 1999).

Further findings on the use of ErwinaseTM within the Scandinavian ALL protocols complement the reports on this product (Albertsen et al, 2001a,b). Doses of 30 000 IU/m2, given i.m. or i.v. during induction, were associated with serum enzyme activities above 500 U/l prior to the next dose in over 90% of cases. When ErwinaseTM was used for reinduction, using two identical doses per week, about 70% of trough levels in the serum were below the threshold of 100 U/l. Depletion of plasma asparagine was observed in about 65% of those samples.

Identical application of different native asparaginases and outcome

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

The clinico-pharmacological findings outlined above are fairly consistent in spite of the differences in analytical and clinical conditions. They gained added importance, as they were subject to the conditions of a randomized clinical study on asparaginase that was run within the framework of the ALL protocols of the European Organisation for Research and Treatment of Cancer (EORTC) (Vilmer et al, 2000; Duval et al, 2002). This study focused on the clinical aspects of efficacy and toxicity of different asparaginase preparations, comparing the E. coli preparations ParonalTM (Christiaens, Breda, The Netherlands; produced by Kyowa Hakko, Tokyo, Japan) and KidrolaseTM (Bellon, Moutrouge, France; produced by Kyowa Hakko, Tokyo, Japan) with the Erwinia chrysanthemi product ErwinaseTM. A total of more than 300 patients per treatment arm were included in the study, with a median follow-up time of 6·9 years. The dosage scheme was similar to the one employed by ALL-BFM. Significant differences between the two treatment arms were observed with regard to both toxicity and efficacy. As to toxicity, the E. coli group showed a higher rate of coagulation disorders. However, the percentage of patients surviving 6 years event-free was also significantly higher in this group (73%) than in the Erwinia chrysanthemi group (60%).

The results of the EORTC studies complement earlier clinico-pharmacological studies in a plausible manner and clearly indicate a close relationship between the therapeutic effect and the treatment intensity achieved by different asparaginase preparations. Moreover, they give evidence that the application of the CrasnitinTM experience-based therapy schemes to all asparaginases in an identical manner will lead to different, possibly limited, outcomes. The evidence supports demands that the product-specific PK properties are to be considered when establishing dosage schemes; it further confirms that, despite all existing uncertainties, realizing a dose intensity comparable with the empirically optimized treatment schemes, verified by records on serum activity and CSF depletion, is the most reliable basis for decisions regarding treatment with different asparaginase preparations at present.

Summary

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References
  • The clinical efficacy of native asparaginases has been proven by single drug studies.
  • Dosage schemes for native asparaginases are based on studies on the E. coli preparation CrasnitinTM.
  • Native asparaginases from different bacterial sources and different native asparaginase preparations from one single source exhibit specific PK profiles.
  • As a consequence, native asparaginases are not readily interchangeable in terms of a uniformly applicable ‘asparaginase dosage recommendation’.
  • Comparable treatment intensity may be achieved by adjusting the application schemes to the product-specific PK properties.
  • Table I summarizes published monitoring data on different dosage schemes for native asparaginases.
Table I.  Referenced overview of findings on pharmacological surrogate parameters in patients undergoing native asparaginase therapy.
ReferenceAsparaginaseDose (IU/m2)ScheduleAsparaginase trough activity (mean ± SD)Asparagine concentration
PlasmaCSF
  1. CSF, cerebrospinal fluid; ASN, asparagine.

Boos et al (1996)Escherichia coli; CrasnitinTM10 000Every 3 d, eight i.v. doses81 ± 51 U/l; 38 samples≤0·1 μmol/l in 33/50 trough samples (60%)
E. coli; MedacTM10 000Every 3 d, eight i.v. doses430 ± 300 U/l; 156 samples≤0·1 μmol/l in 148/169 trough samples (88%)
E. coli; CrasnitinTM10 000Twice weekly, four i.v. doses135 ± 135 U/l; 21 samples≤0·1 μmol/l in 12/21 trough samples (57%)
E. coli; MedacTM10 000Twice weekly, four i.v. doses542 ± 243 U/l; 77 samples≤0·1 μmol/l in 78/86 trough samples (91%)
Erwinia; ErwinaseTM10 000Twice weekly, four i.v. doses<20 U/l; 41 samples≤0·1 μmol/l in 11/49 trough samples (22%)
Gentili et al (1996)Erwinia; ErwinaseTM10 000Every 3 d, eight doses<1 μg/ml in 36/46 trough samples (78%)198 ± 141 ng/ml (mean ± SD); when plasma ASN <1 μg/ml
Erwinia; ErwinaseTM10 000Twice weekly, four doses<1 μg/ml in 3/14 trough samples (21%) 
Ahlke et al (1997)E. coli; MedacTM5000Every 3 d, eight i.v. doses270 ± 109 U/l; 73 samples≤0·1 μmol/l in 70/73 trough samples (96%)
E. coli; MedacTM2500Every 3 d, eight i.v. doses125 ± 68 U/l; 111 samples≤0·1 μmol/l in 108/111 trough samples (97%)<0·1 μmol/l in 18/18 samples from days 29 or 33 of protocol
Vieira Pinheiro et al (1999)Erwinia; ErwinaseTM20 000Mon, Wed, Fri; nine i.v. doses156 ± 99 U/l; day 2; 108 samples<0·2 μmol/l in 30/50 trough samples (60%)
   50 ± 39 U/l; day 3; 51 samplesWith <100 U/l enzyme activity
Rizzari et al (2000)E. coli; MedacTM10 000Every 3 d, eight i.v. doses553 ± 340 U/l; 53 samples<0·2 μmol/l in 90/90 samples from days 28 or 44 of protocol
E. coli; MedacTM10 000Every 3 d, eight i.m. doses678 ± 353 U/l; 37 samples 
Erwinia; ErwinaseTM10 000Every 3 d, eight i.v. doses150 ± 176 U/l; 16 samples<0·2 μmol/l in 36/36 samples from days 28 or 44 of protocol
Erwinia; ErwinaseTM10 000Every 3 d, eight i.m. doses130 ± 120 U/l; 20 samples 
Albertsen et al (2001b)Erwinia; ErwinaseTM30 000Every day, 10 i.v. doses2360 ± 1580 U/l; 51 samples≤0·2 μmol/l in 23/34 trough samples (68%) with <100 U/l enzyme activity
Erwinia; ErwinaseTM30 000Every day, 10 i.m. doses1710 ± 1025 U/l; 93 samples 
Erwinia; ErwinaseTM30 000Mon, Thu; four i.v. doses110 ± 190 U/l; 17 samples 
Erwinia; ErwinaseTM30 000Mon, Thu; four i.m. doses83 ± 189 U/l; 30 samples 

Basic conditions and pharmacology

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

Pegylation, i.e. the covalent binding of PEG molecules to therapeutically applicable proteins, is a common method of the pharmaceutical chemistry that is primarily used to reduce the immunogenic potential of the proteins (e.g. Schellekens, 2002; Harris & Chess, 2003). Moreover, the modification of the molecular protein structure entails essential changes of the PK properties. In the case of PEG asparaginase, these changes translated to an extended half-life of enzyme activity in the serum (Ho et al, 1986; Asselin et al, 1993; Berg et al, 1993), which – compared with the native E. coli-derived original enzyme – was approximately quadrupled in paediatric ALL patients (Asselin et al, 1993).

As a consequence, the pegylated enzyme can be given at a lower dosage and longer intervals when substituted for the native enzyme. From a clinician's point of view, patients will have two basic advantages from this. First, it is expected that the asparaginase-related therapy load may be reduced owing to the lower number of doses. Secondly, exposure to lower amounts of foreign bacterial protein may be expected to produce a reduced rate of hypersensitivity reactions (Kamisaki et al, 1981; Park et al, 1981).

Following the approval of PEG asparaginase in the early 1990s, its introduction into established asparaginase-based treatment strategies was hampered by a relative lack of data supporting recommendations on the ‘proper way of use’. While first studies on the efficacy of PEG asparaginase in patients with relapsed ALL did in fact produce results, which indicated comparability with the native enzymes (Ettinger et al, 1995; review: Holle, 1997), the non-randomized design and small number of patients prevented those findings from gaining evidential status.

Dose finding and PK drug monitoring

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

The introduction of the PEG asparaginase OncasparTM (Medac, Hamburg, Germany; produced by Kyowa Hakko, Tokyo, Japan) into the paediatric BFM protocols for the treatment of newly diagnosed (ALL-BFM) or relapsed ALL (ALL-REZ BFM) was backed up by a drug monitoring programme measuring enzyme activity in the serum. The objective of a treatment intensity comparable with that of native preparations in all treatment phases was approached by single application of doses varying between 500 and 2500 IU/m2 within a drug monitoring programme (Müller et al, 2000, 2002; Vieira Pinheiro et al, 2001, 2002). The main conclusions from the evaluation of c. 1700 values of more than 350 applications can be summarized as follows.

  • In general, the treatment intensity reached was considered comparable with that expected from the application of the native asparaginase. This statement must, however, be qualified in that a relevant number of patients did not even achieve the desired effect. For example, 22 of 66 patients, who received 1000 IU/m2 PEG asparaginase for reinduction under the ALL-BFM protocol, did not show any serum enzyme activity above the threshold of 100 U/l, 2 weeks after the application (Müller et al, 2000).
  • Similarly high inter-individual variability was observed with the application of PEG asparaginase for relapse therapy. However, the monitoring programme produced no results that indicated a relationship between the extent of previous treatment with native preparations or any concurrent clinically manifest hypersensitivity reaction and changes in the activity course of PEG asparaginase over time (Vieira Pinheiro et al, 2001, 2002).
  • Repeated application of PEG asparaginase within several treatment cycles of the ALL-REZ protocol was not associated with any systematic loss of enzyme activity. Prior to a possible ‘silent inactivation’ the intra-individual activity courses over time were fairly easily reproduced (Vieira Pinheiro et al, 2001).
  • Increasing the PEG asparaginase dose did not lead to a correspondingly longer duration of exposure, i.e. the period when the serum activity was above 100 U/l. Irrespective of the dose, the medians of asparaginase activity fell below the target range within 3 weeks following application (Vieira Pinheiro et al, 2002). From a pharmacological point of view, the findings may be interpreted to indicate that the elimination kinetics are insufficiently described by a suggested first-order model (Asselin et al, 1993; Avramis & Holcenberg, 2002). In terms of clinical practice, the findings clearly indicate that repeat application rather than increased dosage might serve to prolong the exposure time.
  • Owing to the highly variable time point of ‘silent inactivation’, observing the course of pharmacological surrogate parameters over time may be most relevant, as it is the only way to identify this phenomenon early on and consequently assure treatment intensity.

Efficacy and PD drug monitoring

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

As with the other asparaginases, studies confirming the relevance of a ‘proper way of use’ are also available for PEG asparaginase; randomized allocation of weekly versus two-weekly application was compared in paediatric patients with ALL relapse (Abshire et al, 2000). Looking at remission as the end point, the group receiving 2500 IU/m2 i.m. on the weekly schedule showed a significantly higher remission rate.

The front-line application of PEG asparaginases has recently been substantiated by efficacy data from two randomized clinical studies. The Dana-Farber Cancer Institute ALL Consortium compared PEG asparaginase 2500 IU/m2 i.m. every other week for 15 doses and E. coli asparaginase 25 000 IU/m2 i.m. every week for 30 doses during the intensification phase of a complex, multiagent regimen, as part of multiple randomizations (Silverman et al, 2001). Event-free survival at 5 years did not differ significantly and was reported to be 84 ± 4% for 92 native asparaginase- and 84 ± 4% for 104 PEG asparaginase-treated patients.

In another study, run in association with the paediatric ALL protocols of the Children's Cancer Group, the single application of 2500 IU/m2 i.m. PEG asparaginase was compared with repeated doses of 6000 IU/m2 i.m. of the native E. coli preparation in c. 60 patients per treatment arm (Avramis et al, 2002). Both groups showed an event-free survival of 3 years in c. 85% of patients. Those data must, however, be interpreted with caution, because the primary end point of this study was the incidence of antibody formation, and the calculation of case numbers, on which the efficacy data are based, had been carried out with a view to this primary end point. Further results on the interplay of PK and PD within the same study give the first indications that the pegylated asparaginase was used in a suboptimal manner. Whereas asparaginase activity in the serum was, in fact, found to stay above 100 U/l for a period of 3 weeks or longer, there was no concurrent complete asparagine depletion in the serum and CSF; asparagine values ranged up to 3 μmol/l. Irrespective of the level of asparaginase activity in the serum, mean values around 1 μmol/l asparagine were found in the serum and CSF. The published report on this study, which focused on issues other than the pharmacology of PEG asparaginase, gives no details on those values. They must, however, be regarded as critical, as they document the existing vagueness regarding the ‘proper way of use’ of PEG asparaginase and also cast doubts on the PK–PD concept which had proven reliable for the native asparaginase; so far, the activities described had always been associated with markedly lower asparagine concentrations and CSF depletion (Boos et al, 1996; Ahlke et al, 1997; Vieira Pinheiro et al, 1999; Rizzari et al, 2000). On the contrary, one should be aware that the pegylated asparaginases used in Europe and in the USA differ in the original E. coli source (Avramis & Holcenberg, 2002); caution is thus advised in interpreting those studies which may have employed biologically non-identical preparations. This is particularly true for particular aspects, which may be explained by differences in PK variables such as kM or Vmax.

Another, related contribution in the current discussion on PEG asparaginase is based on data from a window study associated with the Dutch ALL protocols (Appel et al, 2003). This study investigated the course of asparaginase activity in the serum and asparagine concentration in the CSF after a single i.v. dose of 1000 IU/m2 of the PEG asparaginase preparation OncasparTM. As expected, the enzyme activity stayed above 100 U/l over a minimum of 10 d and was associated with complete peripheral asparagine depletion (<0·2 μmol/l). While asparagine in the CSF was also found to be reduced (e.g. 1·7 μmol/l on day 5 after application), none of the samples were totally depleted.

Summarizing the observations under the application of PEG asparaginase according to two different schemes, it can be stated that asparagine in the CSF remains within a range associated with the continued proliferation of leukaemic blasts in vitro (Cooney & Handschumacher, 1970). The value of asparaginase in the treatment of a possible leukaemic involvement of the central nervous system (CNS) has not been clarified yet. From a theoretical, pharmacological point of view, asparaginase might be postulated as the most active cytotoxic agent for CNS treatment, because the time of continuous exposure to the effect of this substance is longest for leukaemic CNS blasts. Further clinical studies on PEG asparaginase are warranted for a better characterization of the PK–PD profile and for appropriately ranking the pegylated enzyme among the asparaginase armamentarium.

Summary

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References
  • PEG asparaginase has a significantly longer half-life than the native asparaginase.
  • The pegylated enzyme, at lower dosage and longer intervals, can be substituted for native asparaginases.
  • PEG asparaginase, when used as front-line therapy to replace native asparaginases as part of combination chemotherapy, shows comparable efficacy.
  • A dose increase is not associated with a significantly prolonged time of asparaginase activity in the serum above 100 U/l.
  • From a pharmacological point of view, it is preferable with add another dose of PEG asparaginase rather than increasing the dosage when long-term treatment is intended.
  • Two different dosage schemes have been unable to achieve the PD objective of complete asparagine depletion in the CNS.
  • Table II summarizes published monitoring data on different dosage schemes for PEG asparaginases.
Table II.  Referenced overview of findings on pharmacological surrogate parameters in patients undergoing polyethylene glycol asparaginase (PEG-ASNase) therapy.
ReferenceAsparaginaseDose (IU/m2)ScheduleTime after administrationAsparaginase activity, median (range)Asparagine concentration
PlasmaCSF
  1. CSF, cerebrospinal fluid.

Müller et al (2000)PEG-ASNase OncasparTM1000Single i.v. dose; reinductionDay 7/8677 (<20–1085); 61 samples
Day 13 ± 1232 (<20–811) 60 samples
Day 21 ± 138 (<20–214) 55 samples
Vieira Pinheiro et al (2001)PEG-ASNase OncasparTM500Single i.v. dose; relapseDay 0/1413 (<20–693); 53 samples
Day 7 ± 1199 (<20–421); 41 samples
Day 14 ± 1105 (<20–188); 19 samples
Müller et al (2002)PEG-ASNase OncasparTM2500Single i.v. dose; reinductionDay 7 ± 11383 (<20–1997); 37 samples
Day 14 ± 1222 (<20–929); 34 samples
Day 21 ± 1<20 (<20–184); 29 samples
Müller et al (2002)PEG-ASNase OncasparTM2500Single i.v. dose; relapseDay 7 ± 11331 (<20–1985); 17 samples
Day 14 ± 169 (<20–454); 11 samples
Day 21 ± 1<20 (<20–66); 11 samples
Avramis et al (2002)PEG-ASNase2500Single i.m. dose; inductionDay 25>0·03 U/ml in 48% of patients<3 μmol/l when ASNase activity >0·1 U/ml0·6 μmol/l (median)
Appel et al (2003)PEG-ASNase OncasparTM1000Single i.v. dose; window studyDay 0744 ± 132 U/l (mean ± SD)<0·2 μmol/l in 24/24 patients
Day 5483 ± 101 IU/l (mean ± SD) 1·58 ± 0·66 μmol/l (mean ± SD)
Day 12212 ± 66 IU/l (mean ± SD) 
Day 19 2·2 ± 0·67 μmol/l (mean ± SD)
Day 2039 ± 28 IU/l (mean ± SD) 

Future prospects

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References

The described aspects may inspire a number of projects which might contribute to further conclusions on the ‘proper way of use’ of asparaginase.

The application of asparaginase in various treatment protocols has been characterized by high variability due to the use of different drug products with different dosage and application schemes. Information on the PK and PD profile is therefore required in order to evaluate the treatment intensity achieved in a given situation. Continued systematic measurement of the surrogates of asparaginase activity and asparagine concentration or the integration of monitoring programmes in the treatment protocols may produce relevant findings.

Considering the most recent findings on CSF asparagine, further studies of PEG asparaginase with respect to the interplay between PK and PD are needed to better assess its comparability with native asparaginases.

Investigating the immune reaction, which may occur towards any and all asparaginase preparations, may also be considered highly relevant for clinical practice. First results in this segment of research on the development and evaluation of antibody assays (Woo et al, 1998; Wang et al, 2000; Albertsen et al, 2002; Avramis et al, 2002; Wang et al, 2003) stimulate hopes that this tool may be useful in finding the ‘proper way of use’ of asparaginase.

Painstaking documentation of the asparaginase treatment actually given to the patients and the measurement of the resulting dose intensity in terms of serum activity and depletion of asparagine from CSF and plasma are other important tools; they are to be used wherever possible in order to assure the quality of application in the individual case. Study groups, particularly those that employ new asparaginase preparations, have easy access to those methods and are urged to utilize them in order to optimize their treatment schemes and verify the clinico-pharmacological hypotheses behind each dose recommendation.

References

  1. Top of page
  2. Conceptual frame for a rational use of asparaginase
  3. Mechanism of action
  4. PK profile of asparaginase preparations available for clinical application
  5. Relationship between PK and PD
  6. Summary
  7. Native asparaginases
  8. Single drug application
  9. Dose finding studies
  10. Pharmacological surrogates and drug monitoring
  11. Identical application of different native asparaginases and outcome
  12. Summary
  13. PEG asparaginase
  14. Basic conditions and pharmacology
  15. Dose finding and PK drug monitoring
  16. Efficacy and PD drug monitoring
  17. Summary
  18. Future prospects
  19. References
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