ONCH - 1928 - Recent Developments in L-Asparaginase Discovery and Its Potential As Anticancer Agent

ONCH-1928; No.
of Pages 12
ARTICLE IN PRESS
Critical Reviews in Oncology/Hematology xxx (2015) xxxxxx
Recent developments in l-asparaginase discovery and its potential as

anticancer agent
Abhinav Shrivastava a,1 , Abdul Arif Khan a,b,,1 , Mohsin Khurshid b,c , Mohd Abul Kalam b ,
Sudhir K. Jain d , Pradeep K. Singhal e
a College of Life Sciences, Cancer Hospital & Research Institute, Gwalior, MP 474009, India
Department of Pharmaceutics, College of Pharmacy, PO Box 2457, King Saud University, Riyadh 11451, Saudi Arabia
College of Allied Health Professionals, Directorate of Medical Sciences, Government College University, Faisalabad, Pakistan
d Department of Microbiology, Vikram University, Ujjain, MP, India
e Department of Biological Sciences, Rani Durgavati University, Jabalpur, MP, India
b
Accepted 5 January 2015
Contents
1.
2.
3.
4.
5.
6.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mechanism of l-asparaginase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Current asparaginase formulations: a comparative evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Current asparaginase formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1. Native E. coli asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2. Erwinia asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3. PEG-asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Problems associated with clinical application of bacterial asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Hypersensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Coagulation disorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Pancreatitis, hyperglycaemia, hepatotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Resistance to l-asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Advancement in l-asparaginase discovery from alternative sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Algal asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Plants asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Fungal asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Actinomycetes asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5. Entrapment of l-asparaginase in erythrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion and future direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
l-Asparaginase (EC3.5.1.1) is an enzyme, which is used for treatment of acute lymphoblastic leukaemia (ALL) and other related blood
cancers from a long time. This enzyme selectively hydrolyzes the extracellular amino acid l-asparagine into l-aspartate and ammonia, leading to
Corresponding author at: Department of Pharmaceutics, College of Pharmacy, PO Box 2457, King Saud University, Riyadh 11451, Saudi Arabia.
Tel.: +966 542854355.
E-mail address: abdularifkhan@gmail.com (A.A. Khan).
1 These authors have equal contribution.
http://dx.doi.org/10.1016/j.critrevonc.2015.01.002
1040-8428/ 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Shrivastava A, et al. Recent developments in l-asparaginase discovery and its potential as anticancer
agent. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.01.002
ONCH-1928; No. of Pages 12
ARTICLE IN PRESS
A. Shrivastava et al. / Critical Reviews in Oncology/Hematology xxx (2015) xxxxxx
nutritional deficiencies, protein synthesis inhibition, and ultimately death of lymphoblastic cells by apoptosis. Currently, bacterial asparaginases
are used for treatment purpose but offers scepticism due to a number of toxicities, including thrombosis, pancreatitis, hyperglycemia, and
hepatotoxicity. Resistance towards bacterial asparaginase is another major disadvantage during cancer management. This situation attracted
attention of researchers towards alternative sources of l-asparaginase, including plants and fungi. Present article discusses about potential of
l-asparaginase as an anticancer agent, its mechanism of action, and adverse effects related to current asparaginase formulations. This article
also provides an outlook for recent developments in l-asparaginase discovery from alternative sources and their potential as a less toxic
alternative to current formulations.
2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: l-Asparaginase; Acute lymphoblastic leukaemia; Anticancer; l-Asparagine; Erwinase; Kidrolase; PEG-asparaginase
1. Introduction
l-Asparaginase is an important chemotherapeutic agent
for management of acute lymphoblastic leukaemia (ALL)
and other hematopoietic malignancies. In the early fifties,
Kidd identified that guinea pig serum has the ability to control the progression of murine lymphoma. The mice were
transplanted with lymphoma cells during this experiment
and repeated intraperitoneal injections of normal guinea pig
serum were given to them. This treatment led to a regression of lymphoma and survival of treated mice while control
mice grew lymphoma progressively and died within 2030
days [1]. Much earlier the discovery of guinea pig serum
anti-lymphoma activity, it was observed by Clementi (1922),
that guinea pig serum is also a rich source of l-asparaginase
[2]. In the early sixties, guinea pig serum susceptible lymphoma cells were grown in cell culture media devoid of the
amino acid l-asparagine, this inadequacy declined cell population rapidly, but some cells survived and began to proliferate
under l-aspargine limitation. These cells were found to have
lost guinea pig serum susceptibility after transplantation in
mice, while original lymphoma cells retained this susceptibility even after transplantation in mice. The lost susceptibility
to guinea pig serum was restricted to l-asparagine limitation,
while other amino acids, purines and pyrimidines were unable
to complement this effect. Therefore, it was concluded that lasparaginase present in guinea pig serum is responsible for its
antilymphoma activity [3]. These observations also attracted
other researchers for the detection of anticancer potential of
this enzyme, and it was found that the only guinea pig serum
has anti-lymphoma activity, while sera from other animals,
like horse serum and rabbit serum were not able to show comparable activity [1]. Furthermore, the same sera was tested
against many cancer types and observed that it is effective
against only some cancer but not all. Guinea pig serum was
able to inhibit certain lymphoma cell lines in vivo, but the
same activity was not possible with in vitro assays, provided
that guinea pig serum is also a rich source of complement
and proposed anticancer activity against cell lines may be
due to complement mediated cytotoxicity. l-Asparaginase is
an inhibitor of complement in whole guinea pig serum during in vitro assay [4]. Broome performed research to find out
the relative contribution of complement and l-asparaginase
in anticancer activity, and concluded that l-asparaginase is

a major anticancer agent in guinea pig serum [3,5]. The first
clinical trial on l-asparaginase was performed in 1966 by
Dolowy [6]. It is a major chemotherapeutic agent due to
its ability for hydrolysis of l-asparagine into l-aspartic acid
and ammonia. Current asparaginase formulations are used
for management of acute lymphoblastic leukaemia from a
long time, but these formulations are not free from adverse
reaction. Therefore the search for alternative sources of this
enzyme with reduced or no adverse reaction is an important
goal for researchers. This article discusses the potential of
l-asparaginase enzyme as anticancer agents, its mechanism
of action, problems associated with bacterial asparaginases,
future prospects in l-asparaginase discovery and their application as a less toxic alternative.
2. Mechanism of l-asparaginase activity

Clinically used l-asparaginase enzyme from E. coli is
active as a tetrameric protein with identical subunits, each
with a molecular weight of 35.6 kDa according to x-ray
crystallographic data [7]. The molecular formula for each
subunit is C1377 H2208 N382 O442 S17. Chemically it is known
as E. coli l-asparagine amidohydrolase. It is widely used
for the treatment of haemopoietic diseases such as ALL in
children that results from the monoclonal proliferation and
expansion of lymphoid blasts in the bone marrows, blood,
and other organs. ALL correspond to the most common childhood acute leukaemia contributing to approximately 80% of
childhood leukaemias and 20% of adult leukaemias [8]. The
antineoplastic activity results from depletion of the circulating pool of l-asparagine by l-asparaginase, which in turn
inhibits the protein synthesis, causes cell cycle arrest in the
G1-phase and ultimately apoptosis in susceptible leukemic
cells [9,10]. Unlike normal cells, leukemic cells and other
cancer cells have little or no asparagine synthetase and hence
they are not able to carry out the de novo asparagine synthesis, resulting in inhibition of protein synthesis and subsequent
death of the tumour cells [11] (Fig. 1).
Normal cells are protected from asparagine requirement
due to their ability to produce this amino acid [12]. Unlike
conventional cancer therapy, l-asparaginase therapy is highly
ARTICLE IN PRESS
Fig. 1. General mechanism behind selective toxicity of l-asparaginase.
discriminatory. The main restrictions in using l-asparaginase

as a remedial agent is its premature inactivation, more rapid
plasma clearance and shorter duration of drug effect, thus frequent injections are required to maintain a therapeutic level,
and a number of side effects like allergies, development of
immune responses and finally anaphylactic shock, that might
be serious and life-threatening [1315].
The above-mentioned side effects of l-asparaginase may
be due to several reasons, including its l-glutaminase activity.
l-Glutaminase activity of enzyme results in some reduction of plasma l-glutamine level [1618]. l-Asparaginase
is enzymatically active against glutamine, but with a significantly lower affinity against glutamine than l-asparagine.
As glutamine acts as an amino group donor for the enzyme
l-asparagine synthetase for de novo biosynthesis of lasparagine, therefore, the decreased glutamine level because
of l-asparaginase exposure also help in sustaining the
asparagine level reduction and thus contributes the therapeutic effect of l-asparaginase [19] which can be explained as:
l-asparagine
l-asparaginase
l-aspartic acid + NH3
l-glutamine
l-asparaginase
l-glutamic acid + NH3
De novo biosynthesis:
l-aspartic acid + NH3 (From glutamine)
Asparagine Synthetase
l-asparagine
Leukaemia cell after l-asparaginase:

Asparagine No l-asparagine
No Protein Synthesis Cell Death
The exact mechanism of l-asparaginase action is still
not clear, although hydrolysis of l-asparagine is known
to proceed in two steps via a -acyl-enzyme intermediate
(Fig. 2). However, the l-glutaminase activity of asparaginases
contributes to the associated side effects, but this activity
is also related with cell growth inhibition in certain cancer treatments [20,21]. l-Asparaginase induced reduction of
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Fig. 2. Structural illustration of the of l-asparaginase reaction mechanism. During l-asparaginase reaction, covalent intermediate (-Acyl-Enzyme) is formed
through nucleophilic attack by the enzyme. Green arrows indicate a nucleophilic attack. This intermediate later convert into l-aspartate and gives final reaction
products as l-aspratate and ammonia.
asparagine and glutamine level is linked with mTOR pathway and its subsequent inhibition. This pathway includes
an intracellular molecule mammalian target of rapamycin
(known as mTOR). Inhibition of mTOR leads to inhibition
of downstream events in its pathway including phosphorylation of the protein serine threonine kinase (p70S6 kinase
or p70s6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) which suppresses synthesis of ribosomal
proteins at mRNA translation level [22]. It has been found
that inhibition of mTOR pathway by l-asparaginase greatly
affect leukaemia cells and contributes to its antileukaemic
activity. In a separate study on acute myeloid leukaemia, it
has been found that l-asparaginase induces an autophagic
process through inhibition of mTORC1 (mTOR has two
structurally distinct complexes mTORC1 and mTORC2).
mTORC1 is an important regulator of cell growth which activates protein translation and inhibit autophagy under amino
acid abundance by regulating the autophagy related proteins Agt1/ULK and ATG13 [23]. Therefore l-asparaginase
induced reduction of amino acid level is associated with cellular signalling which in turn leads to death of leukeamic
cells.
3. Current asparaginase formulations:
a comparative evaluation
ALL and other lymphoid malignancies have been effectively treated with asparaginase for many years. The
anti-leukemic activity of asparaginases depends upon the
following factors. (i) The rate of hydrolysis, and the Km
of the asparaginase. (ii) The pharmacological factors of
serum clearance of the enzyme [24]. (iii) The development
of asparaginase resistance in tumour cells [18]. (iv) Development of anti-asparaginase antibodies by the host immune
system [25]. (v) The increased contribution of asparagine
either from de novo biosynthesis of asparagine within the
liver or the input from the nutrient intake [26]. The best possible formulation and dosage of asparaginases is still disputed,
despite its use as a vital drug in all treatment protocols for
ALL.
3.1. Current asparaginase formulations

l-Asparaginases have been found not only in mammals,
but also in birds, plants, bacteria (like Escherichia coli,
Erwinia, Salmonella, Mycobacteria, Pseudomonas, and
Acinetobacter species etc.), and fungi (Aspergillus, Fusarium spp. etc.) [27]. Bacillus licheniformis, a member of the
Bacillus subtilis group has recently assessed for production of
l-asparaginase with low glutaminase activity [28]. Similarly,
moderate halophilic bacteria has recently showed production of l-asparaginase [29]. Although not all asparaginases
possess anti-cancer activity and currently, the only preparations available for medical use are the E. coli, Erwinia
chrysanthemi asparaginases, and their derivatives (Table 1).
In the United States, three asparaginase formulations
are widely used against ALL: native E. coli asparaginase (Elspar ; Merck & Co., Inc., West Point, PA, USA),
its Pegylated form (Oncaspar ; Enzon, Inc., Bridgewater, NJ, USA) and Erwinase , the product from cultures
of Erwinia chrysanthemi (Ipsen-Speywood Pharmaceuticals
Ltd, UK). The latter formulation is approved in UK as second line treatment for patients having hypersensitivity to
the former two forms. These native asparaginase formulations are offered under different brand names; Medac
(Kyowa Hakko, Kogyo, Japan), Crasnitin (Bayer AG,
Leverkusen, Germany), Leunase (Sanofi-Aventis, Paris,
France) Paronal , and Kidrolase etc. in Europe and Asia.
Table 1
Clinical pharmacology of asparaginases with frequently administered doses.
Formulation
Elimination half life
Dosage
Native E. coli
asparaginase
PEG-asparaginase
2630 h
Erwinia asparaginase
16 h
6000 IU/m2 three

times/week
20002500 IU/m2
every 2 or 4 weeks
6000 IU/m2
daily ten doses, then
three doses weekly, or
30000 IU/m2
daily ten doses in
induction
5.57 days
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Table 2
Asparaginase formulations therapy in naive adult patients.
Region
First Line
Second Line
North America, UK,

Australia, New
Zealand
Europe
E. coli-asparaginase
or PEG-asparaginase
or PEG-asparaginase
or PEG-asparaginase
Other countries
or PEG-asparaginase
However some of these are no longer available now and only

referred in the literature [24,30] (Table 2).
3.1.1. Native E. coli asparaginase
The asparaginase has improved event-free survival (EFS)
for ALL from <10% to >80% in the past few years. In
early 1990s, Crasnitin (Bayer AG, Leverkusen, Germany)
was not longer available. Therefore asparaginase formulation
Medac was included in Berlin, Frankfurt, Muenster-ALLnon-Hodgkins lymphoma (BFM-ALL-NHL) protocols,
using the similar treatment regimen. However treatment with
Medac leads to more adverse reactions, especially coagulopathies like haemorrhagic and thrombotic events.
In comparison of Medac versus Crasnitin, significant differences were described despite the fact that both enzymes
were products of E. coli strains. Medac showed significantly
higher biological activity so it was possible to decrease
the dose to 2550% of the dosage of Crasnitin in order
to achieve sufficient asparagine depletion under careful
pharmacokinetic and pharmacodynamic monitoring [31,32].
The former was more effective than crasnitin, not only in
asparagine depletion but also in glutamine depletion. Complete asparagine depletion was attained in more than 90%
of the children in treatment with Medac asparaginase. Large
disparities exist between the various products and half life is
particularly reliant on the preparation. Although there is no
need to adjust the individual dose administered, alteration of
the dosing interval is compulsory, resulting in variation to the
number of dosages [33,34]. In a limited number of patients,
the studies with reduced doses of asparaginase Medac were
carried out because asparaginase activity higher than the therapeutic range may lead to increased toxicity. This is apparent
from studies that complete asparagine depletion in serum
and CSF was accomplished by reducing the induction dose
from the common 10000 IU/m2 to 5000 IU/m2 and even to
2500 IU/m2 administered at 3-day intervals in order to reduce
toxicity associated with highly active Medac asparaginase
[31].
3.1.2. Erwinia asparaginase
Erwinia asparaginase (Erwinase) has been used to treat
patients having allergy to E. coli asparaginases. Erwinase was
licensed for use in patients in Europe and is now available in
many countries [35]. Erwinia asparaginase is superior to the
E. coli preparations with reference to immunogenicity and

less induction of coagulation disorders [36].
The efficacy of Erwinase has been doubted as the activity was significantly lower after the first exposure: a trough
level of 100 U/L was achieved in 33% of patients as compared to 94.5% of patients treated with E. coli asparaginase.
Moreover considerably lower i.e. 26% asparagine depletion
has been found after a second exposure despite of equivalent dosages in contrast to the patients in E. coli group i.e.
92.6% [37,38]. In some similar studies with Erwinase complete asparagine depletion was achieved on day 3 of treatment
in only 26% of patients, whereas in some patients no depletion
was found [32]. Therefore, using Erwinia as replacements
for non-pegylated E. coli asparaginases, a higher dose and
increased frequency of treatment is the foremost step to be
taken.
A further necessary feature in the Erwinia asparaginase
therapy is the route of administration. A slower increase of
asparaginase activity is observed after intramuscular (IM)
administration due to the depot effect while intravenous
(IV) administration results in concomitant elevated peak
plasma levels. Asparaginase activities below the desired level
was less observed after Erwinia asparaginase administration
through IM route as compared to intravenous administration
[39]. On the other hand, no significant differences were found
in mean enzyme activity or frequency of samples showing
complete asparagine depletion after IV or IM administration
of Erwinia asparaginase administered during the induction
phase [37]. Likewise, in patients receiving more intense regimen through IM or IV route, analogous complete asparagine
depletion was found in the induction phase [40]. With regards
to the formation of asparaginase neutralizing antibody, no significant dissimilarities were found among the both routes of
administration as a re-induction regimen [41].
In order to ensure maximum survival benefit in ALL
patients, other asparaginase preparation should be employed
in case of development of anti-asparaginase antibodies to a
particular preparation. The assessment of silent inactivation
cases and the degree of serum asparagine depletion is relatively based on asparaginase level investigation. In brief,
depending upon regulatory factors, practice and availability,
Erwinia asparaginase is a valid second or third-line therapy.
Moreover the optimized use of Erwinia asparaginase necessitates further clinical and pharmacokinetic studies [30].
3.1.3. PEG-asparaginase
Nowadays, the most appropriate approach to enhance the
plasma half-life and trim down the immunogenicity and antigenicity of many therapeutic agents is its covalent coupling
with polyethylene-glycol (PEG) and the process is known
as PEGylation [4244]. PEG-asparaginase is the modified
edition of l-asparaginase formed by the covalent conjugation of E. coli asparaginase to monomethoxypolyethylene
glycol (PEG) resulted in significant pharmacokinetic variations in comparison with the native E. coli formulations
[33,45,46]. PEGylation of proteins and enzymes results in
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Fig. 3. Regional PEG-asparaginase therapy in Naive and relapsed children
covering of few surface sites, thus increasing their molecular

size and enhancing steric-hindrance. PEG-asparaginase has
similar chemical properties like native E. coli l-asparaginase
i.e. reaction temperature of 50 C, isoelectic point at pH 5.0
and optimum activity at pH 7.0 [47]. The currently available
PEG-asparaginase formulation in most of the countries is
the E. coli derived asparaginase, Oncaspar [48]. Within the
PEG-asparaginase products from different countries minor
disparities may be found. PEG-asparaginase from the E. coli
l-asparaginase obtained from Merck, Sharp and Dohme are
manufactured by Enzon in US and marketed as Oncaspar ,
while the product is derived from the Kyowa Hakko native
asparaginase protein in Europe [49,50].
The elimination half life of PEG-asparaginase is five
times longer than the native E. coli preparations and nine
times longer than that of Erwinia i.e. around six days. This
represents an imperative improvement in therapy through
decreasing the number of injections needed to achieve therapeutic efficacy in naive patients [19]. In the USA, the
formulation is being approved as first-line therapy whilst in
Europe as a second-line treatment restricted to known patients
allergic to native asparaginases (Fig. 3). In the cited study the
absorption from the injection site was 1.7 days, whereas the
asparaginase activity detectable for a longer period (elimination half-life; 5.5 days) whereas the peak enzymatic activity
in plasma at 5 days after an intramuscular dose of PEGasparaginase [45].
The route of administration is the main factor, because
maximum amino acid depletion and peak enzyme activity
levels are attained within 5 days after intramuscular administration [18]. This step by step depletion of serum asparagine
and glutamine may allow increased production of hepatic
asparagine synthetase and asparagines, therefore diminishing
cytotoxic effects exerted on the cancer cell. The intravenous
route of administration may become the route of choice

because of extensive use and appraisal in adult and paediatric
ALL patient trials. Intravenous route of PEG-asparaginase
will not only deplete asparagine by achieving rapid peak
levels, but also get rid of painful injections [51].
The dose-dependent reduction of CSF asparagine levels can be observed after l-asparaginase administration
despite the fact that PEG-asparaginase penetrates poorly
into the cerebro-spinal fluid (CSF) [18,52]. An IV dose of
PEG-asparaginase 1000 IU/m2 was found insufficient for
asparagine depletion in the CSF whereas significant exhaustion of asparagine in plasma (<0.2 M) was achieved with the
same dose [52]. Asparagine diminution in the CSF usually
happens after completion depletion of asparagine in plasma
[53]. Intravenous PEG-asparaginase dose of 2500 IU/m2 was
sufficient to reduce CSF asparagine levels to <0.2 M as compared to <0.6 M with the same dose of PEG-asparaginase
intramuscularly [18]. PEG-asparaginase also demonstrated
some l-glutaminase activity as found in native E. coli
asparaginases resulting in reduction of glutamine levels in
plasma [18,54].
The pharmacokinetics of PEG-asparaginase is badly
affected by the antibody formation against the asparaginase portion which leads to significant decrease in half-life
[33,55]. This is suggested that the antibody development
against asparaginase fraction is a result of previous exposure
to asparaginase whereas against PEG moiety may be related
to the use of PEG in cosmetics and food products [56].
It is apparent that the initial rationales of developing
PEG-asparaginase have been accomplished [57]. This modified version of l-asparaginases have been effectively used
in patients since last several years because of longer half
life with a reduction of hypersensitivity reactions and number of doses. Even though, still more useful protocols are
ARTICLE IN PRESS
important and the best possible method should be defined for

the use of l-asparaginase in ALL therapy. The production
of neutralizing anti-asparaginase antibodies may be avoided
by the alternate use of various asparaginase formulations
(Native E. coli asparaginase PEG-asparaginase, Erwinia
asparaginase) during the different phases of ALL therapy.
Furthermore, anti-asparaginase antibodies and asparagine
levels should be monitored strictly to facilitate detection of
silent hypersensitivity. In that case, the change in formulation
of asparaginase may be able to improve its activity [45].
4. Problems associated with clinical application of

bacterial asparaginase
Despite significant advancement in l-asparaginase development, current formulations are not free from limitations.
Major adverse events are related to immune reaction to
asparaginase protein. Furthermore asparagine depletion and
subsequent protein synthesis inhibition also aid in restricting
use of asparaginase.
Allergic reactions are primarily due to anti-asparaginase
antibody in circulation and lead to major reason for
toxicity due to l-asparaginase [18,25]. Such complications exaggerate when asparaginase was given without
proper immune-suppressive therapy. Immunosuppression
with steroid reduces the chances of hypersensitivity through
reduction of high titre antibodies. Production of anti
asparaginase antibody was higher with native asparaginase in comparing to PEG-asparaginase. Clinical symptoms
of hypersensitivity include anaphylaxis, pain, oedema,
Quinckes oedema, urticaria, erythema, rash and pruritis [25].
The incidence of skin reactions was high with intramuscular
(IM) administration compared with intravenous (IV) administration. Generally, it has been observed that hypersensitivity
reactions are found during post-induction phase when lasparaginase is not provided for week or months. There are
many reasons for the low frequency of allergic reactions during remission induction, like there is a delay for generation of
proper immune response due to delay in complement activation and subsequent production of antibodies [26]. Allergic
symptoms might hide by intensive corticosteroids therapy
during induction phase [26]. About 30% patients show silent
hypersensitivity or silent inactivation. During this condition
there is rapid inactivation of asparaginase instead of hypersensitivity reaction which leads to asparagine depletion at a
suboptimal level [25,58,59].
Production of anti-asparaginase antibody is responsible
for resistance in asparaginase therapy and also causes the
high level of asparagines in blood plasma [50]. This condition reduces the therapeutic efficacy of asparaginase in some
cases [60,61]. All these conditions demand the alternative
preparation of asaparaginase for removal or low frequency
of allergic reactions [59]. The toxicities of l-asparaginase
immune suppression may divide into two types; firstly those
that belong to immunologic sensitization to a foreign protein

and another related to the protein synthesis inhibition [62].
4.1. Hypersensitivity
As discussed earlier, bacterial proteins induce immunologic reactions leading to localized, transient erythema
and rash at the site of injection to urticaria, respiratory
distress, and acute life-threatening anaphylaxis. Clinical presentation of asparaginase hypersensitivity reactions may
range from, allergic reactions, anaphylaxis (rare), serum
sickness, itching and swelling of extremities, oedema,
urticaria, rash and broncospasm, localized or generalized
erythema, and other clinically related reactions. Moreover,
asparaginases treatment can also lead to organ toxicities,
liver dysfunction, pancreatitis and related hyperglycemia,
ketoacidosis, glucosuria, cerebral dysfunction, decreased
protein synthesis, hypofibrinonemia, hypercoagulable statecoagulopathies, hypoalbuminemia [26].
It has been observed that about 60% patients were suffering from hypersensitivity reactions during therapy of
l-asparaginase from E. coli. Even E. coli derived PEG
asparaginase also shows hypersensitivity reactions, thus
requiring a shift to another form of asparaginase [63].
4.2. Coagulation disorder
Asparaginase therapy can also lead to several coagulation
related abnormalities due to severe acquired deficiency of
serpin (inhibitor of serine protease) proteins. These proteins
include antithrombin and 1 antitrypsin. Such complications are also higher with native forms of l-asparaginase in
comparing to PEG asparaginase with the risk of 2.115%
and 1.14% respectively [55,64]. Physiological inhibition of
thrombin and other coagulation factor like IXa, Xa, XIa as
well as factor VII is majorly regulated through antithrombin,
which forms an irreversible link with enzyme leading to its
proteolytic activity inhibition [65]. Researchers have identified that conformational changes in antithrombin molecule
induced by l-asparaginase are responsible for the loss of its
activity and formation of protein aggregates accumulated in
ER cisterns [66]. Furthermore, coagulation disorders associated side effects are produced due to the effect of drug
on protein synthesis, reduction in antithrombin, fibrinogen,
plasminogen, and factors IX and X with prolongation of activated partial thromboplastin time [67,68]. l-Asparaginase
treatment can also lead to protein C and S deficiency, which
ultimately increases thrombin level and increase the risk
of bleeding and thrombosis [69,70]. Generally, 1565% of
patients also show hypofibrinogenemia after asparaginase
therapy.
4.3. Pancreatitis, hyperglycaemia, hepatotoxicity
Although, the reason behind such adverse reaction is
not well defined but it has been reported to occur due to
ARTICLE IN PRESS
defects in protein synthesis. Asparaginase treatment associated liver toxicity includes oxidative stress, glutamine
deficiency, decreased hepatic protein synthesis, and later
on impairment of beta-oxidation in mitochondria [7173].
Patients with liver abnormalities through asparaginase treatment rarely show its histological presentation but macro- and
microvesicular liver steatosis have been described [71,74].
Furthermore, l-asparaginase treatment can also increase
pancreatic amylase and lipase. Synthesis inhibition of these
enzymes is regulated by l-asparagine. In the absence of lasparagine, these enzymes create severe complications in
pancreas [65,75]. l-Asparaginase therapy can also induce
diabetes in some patients due to decreased insulin synthesis
through affecting both the endocrine (insulin-secreting) and
exocrine (digestive enzyme-secreting) cells of the pancreas.
In addition to above complications, CNS related
symptoms are another adverse events associated with lasparaginase therapy. It has been proposed that a reduction
in level of l-asparagine and l-glutamine in cerebral tissues is responsible for these adverse reactions. Symptoms
of l-asparaginase associated CNS related adverse events
include hallucination, drowsiness, and amentia [76]. It has
also reported that l-asparaginase therapy is associated with
posterior reversible encephalopathy syndrome (PRES) [77].
4.4. Resistance to l-asparaginase
The mechanisms behind the failure and resistance to lasparaginase treatment is still doubtful [78,79]. Activity of
l-asparaginase is dependent on the development of antil-asparaginase antibodies, leading to failure of asparagine
depletion after re-administration of l-asparaginase, and
causes l-asparaginase resistance [25,51]. These antil-asparaginase antibodies neutralize the l-asparaginase
activity, resulting in more rapid plasma clearance and shorter
duration of drug effect. Antibody development may occur in
patients having clinical allergy symptoms [24,25,32].
Resistance to l-asparaginase therapy may also be assumed
because of the high level of l-asparaginase synthetase cellular expression, that allows the production of asparagine and
hence protein synthesis. Expression of cellular l-asparagine
synthetase is found low in B-lineage ALL with TEL/AML1
and the hyper-diploid subtype and may be an indication of
their increased sensitivity to l-asparaginase [78,80]. Conversely, in some new studies of TEL/AML1 ALL cells,
over expression of asparagine synthetase mRNA was originated and did not correlate with resistance to l-asparagine
in TEL/AML1 [81]. In other words, it can be summarized
that cellular asparaginase synthetase over expression was
shown to be higher in T-lineage ALL and lower in B-lineage
ALL with translocation-erythroblastosis virus E26 oncogene
homolog leukaemia-acute myeloid leukaemia 1 TEL-AML1
fusion gene [78].
Resistance to l-asparaginase also arise from the increased
expression of asparagine synthetase stimulated by the tumour
cells after exposure to l-asparaginase and from the expression
of asparagine synthetase by the mesenchymal cells of the

bone marrow which constitute the marrow microenvironment
for leukaemia cells where the leukemic cells mature [78,79].
Co-culture with mesenchymal cells protected ALL cells from
the cytotoxicity caused by l-asparaginase, and this shielding effect correlated with asparagine synthetase levels, i.e.
down regulation by RNA interference decreased the shielding
effects of mesenchymal cells, whereas enforced asparagine
synthetase expression give enhanced protection. The authors
suggested that mesenchymal cells-mediated niches in the
bone marrow form a safe haven for ALL blasts by increasing
concentrations of asparagine in the leukemic cell microenvironment [78]. It is yet to be decided whether this association
is critical in vivo or if the relationship between the leukemic
cells and mesenchymal cells contributes to development
of minimal residual disease in ALL patients [79]. Other
mechanisms regulating sensitivity of leukemic cells to lasparaginase are still doubtful and unstated. l-Asparaginases
are p53-dependent therapeutic agents that cause cytotoxicity
by the mechanism of apoptosis. Other key apoptotic genes
associated with l-asparaginase resistance are BCL2L13,
Harakiri and TNF [45,82].
5. Advancement in l-asparaginase discovery from

alternative sources
In order to overcome the limitations of current asparaginase formulations, modified preparations of l-asparaginase
have been proposed [83]. Occurrence of l-asparaginases in
fungi, yeasts, bacteria and animal cells, and their antitumour effects were reviewed in many articles [84,85], but the
majority of l-asparaginases cannot be translated for human
applications. Despite this, discovery of l-asparaginase in several organisms raises optimism about the development of this
therapeutic agent with less adverse reactions.
5.1. Algal asparaginase
l-Asparaginase from a Chlamydomonas species has been
purified to near homogeneity (78 units/mg of protein) as
determined by disc-gel electrophoresis. Its molecular weight
and Km were about 275Kd and 1.34 104 M respectively.
The purified enzyme was stable at room temperature for
24 days in sterile solution and showed the greatest activity at 55 C. The Chlamydomonas l-asparaginase possess
little ability to inhibit the growth of the Gardners lymphosarcoma in C3H mice [86]. As an important requirement
for anti-tumour activity, this enzyme showed good activity
at physiological pH and temperature, no inhibition of the
enzyme by reaction end products, no cofactor requirement,
slow rate of clearance from the serum and relatively low
Km [87]. Although, the Chlamydomonas asparaginase has
satisfied many of these requirements, it Km is higher than
asparaginases employed in chemotherapy [85]. Kinetic studies with this enzyme indicate that the relatively high Km
ARTICLE IN PRESS
value may have limited its antitumour activity, although other

factors such as serum survival were not investigated [88].
5.2. Plants asparaginase
In comparison to the bacterial enzymes, the plant enzymes
have been studied less thoroughly. In plants, l-asparagine is
the major nitrogen storage and transport compounds, those
are utilized in protein synthesis in the actively growing tissues. Asparagine is the major nitrogen assimilatory product
of nitrogen fixation and nitrate reduction in Lupinus and many
other legumes [89].
There are two groups of such proteins, called potassiumdependent and potassium-independent asparaginases [90].
The plant asparaginase amino acid sequences did not have
any significant similarity with microbial asparaginase but was
23% identical and 66% similar to a human glycosylasparaginase [89].
Withania somnifera L. has been traditionally used in
various folklores as a sedative and hypnotic. Recently,
in vitro cytotoxicity of l-asparaginase from W. somnifera
was observed in a study where enzyme was purified from
the fruits of W. somnifera L. The antitumour and growth
inhibitory effect of the l-asparaginase was assessed using
[3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl-tetrazolium bromide] (MTT) calorimetric dye reduction method. This
research was proposed as first report of the plant containing lasparaginase with antitumour activity. W. somnifera enzyme
has less toxicity compared to bacterial l-asparaginase [91].
5.3. Fungal asparaginase
A long time ago, researchers found the l-asparaginase
activity in Penicillium camemberti [92]. In 1930, lasparaginase of Aspergillus niger was studied by Schmalfuss
& Mothes [93]. De-Angeli et al. (1970) reported lasparaginase isolated from A. terreus, which suppressed the
growth of Walker 256 ascites sarcoma in rats [94]. Scheetz
et al. (1971) studied the properties of l-asparaginase purified
from mycelia of Fusarium tricinctum, and pointed out the
inability of the enzyme to suppress the growth of GC3HED
lymphosarcoma in mice [95]. Arima et al. (1972) examined
the extracellular formation of l-asparaginase by a variety of
micro-organisms and indicated that the enzyme was produced
by several species of Penicillium, Aspergillus, and Nectria
[96]. l-Asparaginase from Cladosporium cladosporiodies
shows anti-tumour activity against Erlichs ascites in mice
[97].
Recently, Shrivastava et al. (2010) found several fungi
with l-asparaginase activity. Few of them show the cytotoxic
effect against various human cancer cell lines [98,99]. lAsparaginase from Aspergillus niger was also obtained which
show no cytotoxicity against normal human cells, but show
anti-proliferative effect against leukemic cell line RS4; and
HL60 [100].
5.4. Actinomycetes asparaginase

l-Asparaginase with antineoplastic activity is also found
in actinomycetes. Partially purified enzyme with molecular
weight 140Kd , optimum stability at temperature 60 C and
pH 8.0. l-Asparaginase showed a cytostatic effect in 24 h,
whereas the cytotoxic effect in 48 h against JURKAT and
K562 human cancer cell line [101].
5.5. Entrapment of l-asparaginase in erythrocytes
Erythrocytes can be used as a microbioreacter. As investigated by Ataullakhanov et al. (1985), asparagine can
be transferred into human erythrocytes from the external
medium. l-Asparaginase can enter in the erythrocytes by
reversible osmotic lysis [102]. The enzyme remains active
and efficient in the erythrocytes during the circulation flow.
There is no significant alteration of half life of transfused
erythrocytes. The erythrocyte membrane provides protection
to l-asparaginase from anti-l-asparaginase antibodies which
may neutralize or significantly reduce enzyme activity.
Using red blood cells as a micro-bioreactor for enzyme
therapy presents many advantages, especially increased halflife and the reduction in hypersensitivity. Therefore, using red
cells as a new entrapped form would protect enzymes from a
rapid degradation in the blood and provide the safety organism from side effects. Due to prolonged one month half-life,
the low frequency of injections would be more comfortable
for patients with this preparation [103].
In addition to the above mentioned alternative sources,
human glycosylasparaginase is also investigated due to their
potential of l-asparagine hydrolysis into l-aspartate and
ammonia similar to bacterial asparaginases without any
l-glutaminase activity. It is suggested that human glycosylasparaginases can be used to reduce side effects associated
with bacterial asparaginases due to absence of glutaminase
activity [104].
6. Conclusion and future direction

Asparaginase therapy is an important component in the
treatment of children with ALL. However complications may
arise due to certain side effects of the enzyme when used
as a chemotherapeutic agent. The complications may range
from mild hypersensitivity to anaphylactic shocks. The toxic
effects of asparaginase are related primarily to immune reactions to this bacterial protein and to the effects of asparagine
depletion, and its subsequent inhibition of protein synthesis
in the major glands such as the liver and pancreas. The allergic reactions are the most prominent toxicities. Polyethylene
glycol-conjugated asparaginase has significant pharmacological advantages over native Escherichia coli asparaginase.
The enzyme derived from Erwinia carotovora does not share
antigenic cross-reactivity with the E. coli enzyme and, therefore, has been used when patients develop hypersensitivity
10
ARTICLE IN PRESS
to the E. coli enzyme. This condition attracted scientist for

discovering this activity in many other organisms. In fact the
research gathers optimism about prospects of l-asparaginase
obtained from alternative sources as therapeutic agents for
management of ALL and other related malignancies. Perhaps, future research can usher in the development of suitable
alternative of l-asparaginase which can pass all limitations
posed by current asparaginase formulations.
Conict of interest
Authors do not have any personal or financial conflict of
interest related to this work.
Reviewers
Xu Zhen, Institute of Molecular Medicine, Center for
Metabolic and Degenerative Diseases, 1825 Pressler Street,
Houston, TX 77030, United States.
Dr Zakir Khan, Department of Biotechnology, Madhav
Institute of Technology and Science, Gwalior, India.
Acknowledgement
The authors are grateful to Deanship of Scientific Research
and Research Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
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Biographies
Dr. Abhinav Shrivastava is working as Assistant Professor in College of Life Sciences, Cancer Hospital and
Research Institute, Gwalior, India. His primary research
interest includes the development of L-asparaginase from
alternative sources in order to develop less toxic enzyme formulation. He is involved in the isolation of L-asparaginase
from plants, animals, and microorganisms.
Dr. Abdul Arif Khan is an Assistant Professor in College
of Pharmacy, King Saud University, Riyadh, Saudi Arabia.
His current research focus is on cancer associated bacterial
infections and their role in cancer aetiology and diagnosis,
development of anticancer substances from microbial origin,
and cancer system biology.
Mr. Mohsin Khurshid is a Lecturer at Directorate of
Medical Sciences, Government College University, Faisalabad, Pakistan. Previously he has served as Lecturer in
College of Pharmacy, King Saud University, Riyadh, Saudi
Arabia. His research interest includes the bacterial Pathogenesis, antibiotic resistance mechanisms among bacteria
and the potential role of bacteria in cancer diagnosis and
management.
Dr. Mohd Abul Kalam has completed his PhD (Pharmaceutics) in June 2011 from Department of Pharmaceutics,
Faculty of Pharmacy, Jamia Hamdard, New Delhi, India.
Since November 2011, he is working as Assistant Professor at Department of Pharmaceutics, College of Pharmacy,
King Saud University, Riyadh, Saudi Arabia. His research
interest includes the development of nanoformulations as a
novel drug delivery system.
Dr. Sudhir K Jain is working as Associate Professor
in Department of Microbiology. Vikram University, Ujjain,
India. His research activities are focused on identification
of novel drug targets for new drug development, secondary
metabolite production from Keratinophilic and soil fungi.
Dr. Pradeep K Singhal is a Professor in Department of Biological Science, Rani Durgavati University,
Jabalpur, India. His research interest includes environmental
biotechnology and development of anticancer formulations
including L-asparaginase.

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ONCH-1928; No.

Critical Reviews in Oncology/Hematology xxx (2015) xxxxxx

Recent developments in l-asparaginase discovery and its potential as

Accepted 5 January 2015

ONCH-1928; No. of Pages 12

in anticancer activity, and concluded that l-asparaginase is

2. Mechanism of l-asparaginase activity

ONCH-1928; No. of Pages 12

Fig. 1. General mechanism behind selective toxicity of l-asparaginase.

discriminatory. The main restrictions in using l-asparaginase

l-aspartic acid + NH3

l-glutamic acid + NH3

Leukaemia cell after l-asparaginase:

ONCH-1928; No. of Pages 12

3.1. Current asparaginase formulations

Elimination half life

6000 IU/m2 three

ONCH-1928; No. of Pages 12

A. Shrivastava et al. / Critical Reviews in Oncology/Hematology xxx (2015) xxxxxx

North America, UK,

However some of these are no longer available now and only

E. coli preparations with reference to immunogenicity and

ONCH-1928; No. of Pages 12

Fig. 3. Regional PEG-asparaginase therapy in Naive and relapsed children

covering of few surface sites, thus increasing their molecular

route of administration may become the route of choice

ONCH-1928; No. of Pages 12

important and the best possible method should be defined for

4. Problems associated with clinical application of

that belong to immunologic sensitization to a foreign protein

ONCH-1928; No. of Pages 12

A. Shrivastava et al. / Critical Reviews in Oncology/Hematology xxx (2015) xxxxxx

of asparagine synthetase by the mesenchymal cells of the

5. Advancement in l-asparaginase discovery from

ONCH-1928; No. of Pages 12

value may have limited its antitumour activity, although other

5.4. Actinomycetes asparaginase

6. Conclusion and future direction

ONCH-1928; No. of Pages 12

to the E. coli enzyme. This condition attracted scientist for

[9] Lee SM, et al. L-asparaginase from Erwinia carotovora. An improved

ONCH-1928; No. of Pages 12

ONCH-1928; No. of Pages 12

[77] Thachil J. L-Asparaginase, nitric oxide and posterior reversible

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