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Recent developments in -asparaginase discovery and its


potential as anticancer agent Article Tools
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Abhinav Shrivastava1, Abdul Arif Khan1, , Mohsin Khurshid, Mohd Abul Kalam, Sudhir K. Jain, Pradeep
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DOI: http://dx.doi.org/10.1016/j.critrevonc.2015.01.002 |
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1. Introduction
2. Mechanism of l-asparaginase activity
3. Current asparaginase formulations: a comparative evaluation
3.1. Current asparaginase formulations
3.1.1. Native E. coli asparaginase Related Articles
3.1.2. Erwinia asparaginase
3.1.3. PEG-asparaginase
4. Problems associated with clinical application of bacterial asparaginase Pharmacological and clinical evaluation
4.1. Hypersensitivity of -asparaginase in the treatment of
4.2. Coagulation disorder leukemia
4.3. Pancreatitis, hyperglycaemia, hepatotoxicity Critical Reviews in Oncology / Hematology,
4.4. Resistance to l-asparaginase Vol. 61, Issue 3
5. Advancement in l-asparaginase discovery from alternative sources
5.1. Algal asparaginase Use of -asparaginase in childhood ALL
5.2. Plants asparaginase Critical Reviews in Oncology / Hematology,
5.3. Fungal asparaginase Vol. 28, Issue 2
5.4. Actinomycetes asparaginase
5.5. Entrapment of l-asparaginase in erythrocytes
Clinical updates in adult acute
6. Conclusion and future direction
Conflict of interest lymphoblastic leukemia
Reviewers Critical Reviews in Oncology / Hematology,
Acknowledgement Vol. 99
References
Biography Lymphoblastic lymphoma
Critical Reviews in Oncology / Hematology,
Vol. 113
Jump to Section Go
Highlights Central nervous system chemotoxicity
during treatment of pediatric acute
-Asparaginase kills asparagine dependent leukaemia cells by hydrolysing asparagine. lymphoblastic leukemia/lymphoma
Bacterial -asparaginase is used for treatment of leukaemia and other blood cancer. Critical Reviews in Oncology / Hematology,
Bacterial -asparaginase is associated with several adverse reactions. Vol. 84, Issue 2
These adverse reactions necessitate development of -asparaginase from other sources.
View All

Jump to Section Go
Abstract
-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 -asparagine into -aspartate and ammonia, leading to 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 -
asparaginase, including plants and fungi. Present article discusses about potential of -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 -asparaginase discovery from alternative
sources and their potential as a less toxic alternative to current formulations.

Keywords:

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l-Asparaginase, Acute lymphoblastic leukaemia, Anticancer, l-Asparagine, Erwinase, Kidrolase, PEG-
asparaginase

Jump to Section Go
1. Introduction
-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 (Kidd, 1953). 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 -
asparaginase. In the early sixties, guinea pig serum susceptible lymphoma cells were grown in cell culture media
devoid of the amino acid -asparagine, this inadequacy declined cell population rapidly, but some cells survived
and began to proliferate under -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 -asparagine limitation, while
other amino acids, purines and pyrimidines were unable to complement this effect. Therefore, it was concluded
that -asparaginase present in guinea pig serum is responsible for its antilymphoma activity (Broome, 1963).
These observations also attracted other researchers for the detection of anticancer potential of this enzyme, and
it was found that 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 (Kidd, 1953). 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. -Asparaginase is an inhibitor of
complement in whole guinea pig serum during in vitro assay (Delage et al., 1971). Broome performed research to
find out the relative contribution of complement and -asparaginase in anticancer activity, and concluded that -
asparaginase is a major anticancer agent in guinea pig serum (Broome, 1963, Patil et al., 2011). The first clinical
trial on -asparaginase was performed in 1966 by Dolowy (Dolowy et al., 1966). It is a major chemotherapeutic
agent due to its ability for hydrolysis of -asparagine into -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 reactions. 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 -asparaginase
enzyme as anticancer agents, its mechanism of action, problems associated with bacterial asparaginases, future
prospects in -asparaginase discovery and their application as a less toxic alternative.

Jump to Section Go
2. Mechanism of -
asparaginase activity
Clinically used -asparaginase enzyme from E. coli is active as a tetrameric protein with identical subunits, each
with a molecular weight of 35.6kDa according to x-ray crystallographic data (Swain et al., 1993). The molecular
formula for each subunit is C1377H2208N382O442S17. Chemically it is known as E. coli -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 (Fullmer et al., 2010). The antineoplastic activity results from
depletion of the circulating pool of -asparagine by -asparaginase, which in turn inhibits the protein synthesis,
causes cell cycle arrest in the G1-phase and ultimately apoptosis in susceptible leukemic cells (Lee et al., 1989,
Keating et al., 1993). 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 (Killander et al., 1976) (Fig. 1).

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

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Normal cells are protected from asparagine requirement due to their ability to produce this amino acid (Kotzia et
al., 2007). Unlike conventional cancer therapy, -asparaginase therapy is highly discriminatory. The main
restrictions in using -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 (Mashburn and Wriston, 1964, Moola et al., 1994, Soares et al., 2002).

The above-mentioned side effects of -asparaginase may be due to several reasons, including its -glutaminase
activity. -Glutaminase activity of enzyme results in some reduction of plasma -glutamine level (Distasio et al.,

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1982, Villa et al., 1986, Avramis et al., 2002). -Asparaginase is enzymatically active against glutamine, but with
a significantly lower affinity against glutamine than -asparagine. As glutamine acts as an amino group donor for
Targeting autophagy as a
the enzyme -asparagine synthetase for de novo biosynthesis of -asparagine, therefore, the decreased potential therapeutic
glutamine level because of -asparaginase exposure also help in sustaining the asparagine level reduction and approach for immune
thus contributes the therapeutic effect of -asparaginase (Zeidan et al., 2009) which can be explained as: thrombocytopenia therapy
Shan, Ning-ning et al.

De novo biosynthesis:

Leukaemia cell after -asparaginase:

The exact mechanism of -asparaginase action is still not clear, although hydrolysis of -asparagine is known to
proceed in two steps via a -acyl-enzyme intermediate (Fig. 2). However, the -glutaminase activity of
asparaginases contributes to the associated side effects, but this activity is also related with cell growth inhibition
in certain cancer treatments (Wu et al., 1978, Wise and Thompson, 2010). -Asparaginase induced reduction of
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 (Iiboshi et al., 1999). It has been found that inhibition of
mTOR pathway by -asparaginase greatly affect leukaemia cells and contributes to its antileukaemic activity. In a
separate study on acute myeloid leukaemia, it has been found that -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 (Jacque and
Bouscary, 2014). Therefore -asparaginase induced reduction of amino acid level is associated with cellular
signalling which in turn leads to death of leukeamic cells.

Fig. 2
Structural illustration of -asparaginase reaction mechanism. During -asparaginase reaction, covalent
intermediate (-Acyl-Enzyme) is formed through nucleophilic attack by the enzyme. Green arrows indicate
a nucleophilic attack. This intermediate later converts into -aspartate and gives final reaction products as
-aspratate and ammonia.

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Jump to Section Go
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 (Avramis and Panosyan,
2005). (iii) The development of asparaginase resistance in tumour cells (Avramis et al., 2002). (iv) Development
of anti-asparaginase antibodies by the host immune system (Panosyan et al., 2004). (v) The increased
contribution of asparagine either from de novo biosynthesis of asparagine within the liver or the input from the
nutrient intake (Avramis and Tiwari, 2006). 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 Jump to Section Go


formulations
-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.) (Verma et al., 2007). Bacillus licheniformis, a member of the Bacillus subtilis group has
recently assessed for production of -asparaginase with low glutaminase activity (Mahajan et al., 2012). Similarly,
moderate halophilic bacteria has recently showed production of -asparaginase (Ebrahiminezhad et al., 2012).
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).

Table 1
Clinical pharmacology of asparaginases with frequently administered doses.

Elimination
Formulation half life Dosage

Native E. coli 2630h 6000IU/m2 three times/week

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asparaginase

PEG-asparaginase 5.57 days 20002500IU/m2 every 2 or 4 weeks

Erwinia 16h 6000IU/m2 dailyten doses, then three doses weekly, or 30000IU/m2
asparaginase dailyten doses in induction

View Table in HTML

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; Sigma-Tau Pharmaceuticals,
Inc. Gaithersburg, MD, USA) and Erwinaze, the product from cultures of Erwinia chrysanthemi (Jazz
Pharmaceuticals, Palo Alto, CA, USA). The Erwinia formulation is approved in UK as second line treatment for
patients having hypersensitivity to the former two forms with brand name Erwinase (EUSA Pharma,
International division of Jazz Pharmaceuticals). These native asparaginase formulations are offered under
different brand names; Medac (Kyowa Hakko, Kogyo, Japan), Crasnitin (Bayer AG, Leverkusen, Germany),
Leunase, Paronal, and Kidrolase etc. in Europe and Asia. However some of these are no longer available
now and only referred in the literature (Avramis and Panosyan, 2005, Pieters et al., 2010) (Table 2).

Table 2
Asparaginase formulations therapy in naive adult patients.

Region First Line Second Line

North America, UK, Australia, New E. coli-asparaginase or PEG- Erwinia asparaginase or PEG-
Zealand asparaginase asparaginase

Europe E. coli-asparaginase or PEG- Erwinia asparaginase


asparaginase

Other countries E. coli-asparaginase Erwinia asparaginase or PEG-


asparaginase

View Table in HTML

3.1.1. Native E. coli asparaginase Jump to Section Go


The asparaginase has improved event-free survival 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-ALL-non-Hodgkin's 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 (Ahlke et al., 1997, Boos et al., 1996). 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 (Asselin et al., 1993, Muller and Boos, 1998). 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 10000IU/m2 to 5000IU/m2 and even to 2500IU/m2 administered at 3-day intervals in order to
reduce toxicity associated with highly active Medac asparaginase (Ahlke et al., 1997).

3.1.2. Erwinia asparaginase Jump to Section Go


Erwinia asparaginase 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 (Salzer et al., 2012). Erwinia
asparaginase is superior to the E. coli preparations with reference to immunogenicity and less induction of
coagulation disorders (Albertsen et al., 2001a).

The efficacy of Erwinase has been doubted as the activity was significantly lower after the first exposure: a
trough level of 100U/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% (Rizzari et al., 2000,
Gentili et al., 1996). 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 (Boos et al., 1996).
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 (Schrey et al., 2010). 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 (Rizzari et al., 2000). Likewise,
in patients receiving more intense regimen through IM or IV route, analogous complete asparagine depletion was

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found in the induction phase (Albertsen et al., 2001b). However, both IM and IV formulations of Erwinaze are
FDA approved in USA. 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 (Albertsen et al.,
2002).

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 (Pieters et al., 2010).

3.1.3. PEG-asparaginase Jump to Section Go


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 (Veronese, 2001, Veronese and Pasut, 2005, Harris and Chess, 2003). PEG-
asparaginase is the modified edition of -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 (Asselin et al., 1993, Fu and Sakamoto, 2007, Vieira Pinheiro et
al., 2001). PEGylation of proteins and enzymes results in covering of few surface sites, thus increasing their
molecular size and enhancing steric-hindrance. PEG-asparaginase has similar chemical properties like native E.
coli -asparaginase i.e. reaction temperature of 50C, isoelectic point at pH 5.0 and optimum activity at pH 7.0
(Wada et al., 1990). The currently available PEG-asparaginase formulation in most of the countries is the E. coli
derived asparaginase, Oncaspar (Berg, 2010). Within the PEG-asparaginase products from different countries
minor disparities may be found. PEG-asparaginase from the E. coli -asparaginase obtained from Merck, Sharp
and Dohme are manufactured by Sigma-Tau in US and marketed as Oncaspar, while the product is derived
from the Kyowa Hakko native asparaginase protein in Europe (Vieira Pinheiro et al., 2006, Hak et al., 2004).

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 (Zeidan et
al., 2009). 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 PEG-
asparaginase (Fu and Sakamoto, 2007).

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

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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 (Avramis et al., 2002). 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 (Douer et al., 2007).

The dose-dependent reduction of CSF asparagine levels can be observed after -asparaginase administration
despite the fact that PEG-asparaginase penetrates poorly into the cerebro-spinal fluid (CSF) (Avramis et al.,
2002, Appel et al., 2003). An IV dose of PEG-asparaginase 1000IU/m2 was found insufficient for asparagine
depletion in the CSF whereas significant exhaustion of asparagine in plasma (<0.2M) was achieved with the
same dose (Appel et al., 2003). Asparagine diminution in the CSF usually happens after completion depletion of
asparagine in plasma (Riccardi et al., 1981). Intravenous PEG-asparaginase dose of 2500IU/m2 was sufficient to
reduce CSF asparagine levels to <0.2M as compared to <0.6M with the same dose of PEG-asparaginase
intramuscularly (Avramis et al., 2002). PEG-asparaginase also demonstrated some -glutaminase activity as
found in native E. coli asparaginases resulting in reduction of glutamine levels in plasma (Avramis et al., 2002,
Miller et al., 1969).

The pharmacokinetics of PEG-asparaginase is badly affected by the antibody formation against the
asparaginase portion which leads to significant decrease in half-life (Asselin et al., 1993, Holle, 1997). 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
(Armstrong et al., 2007).

It is apparent that the initial rationales of developing PEG-asparaginase have been accomplished (Graham,
2003). This modified version of -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 important and the best possible method should be defined for the use of -

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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 (Fu and Sakamoto, 2007).

Jump to Section Go
4. Problems associated
with clinical application
of bacterial asparaginase
Despite significant advancement in -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 -asparaginase (Avramis et al., 2002, Panosyan et al., 2004). 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, Quincke's oedema, urticaria, erythema, rash
and pruritis (Panosyan et al., 2004). 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 -asparaginase is not provided for weeks
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 (Avramis and Tiwari, 2006). Allergic symptoms might hide by intensive
corticosteroids therapy during induction phase (Avramis and Tiwari, 2006). 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 (Panosyan et al., 2004,
Asselin, 1999, Wenner et al., 2005).

Production of anti-asparaginase antibody is responsible for resistance in asparaginase therapy and also causes
the high level of asparagines in blood plasma (Hak et al., 2004). This condition reduces the therapeutic efficacy
of asparaginase in some cases (Larson et al., 1998, Woo et al., 2000). All these conditions demand the
alternative preparation of asaparaginase for removal or low frequency of allergic reactions (Wenner et al., 2005).
The toxicities of -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 (Wang et al.,
2003).

4.1. Hypersensitivity Jump to Section Go


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 state-coagulopathies,
hypoalbuminemia (Avramis and Tiwari, 2006).

It has been observed that about 60% patients were suffering from hypersensitivity reactions during therapy of -
asparaginase from E. coli. Even E. coli derived PEG asparaginase also shows hypersensitivity reactions, thus
requiring a shift to another form of asparaginase (Pieters et al., 2011).

4.2. Coagulation disorder Jump to Section Go


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 -asparaginase in comparing to PEG
asparaginase with the risk of 2.115% and 1.14% respectively (Holle, 1997, Nowak-Gottl et al., 2003).
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 (Piatkowska-Jakubas et al., 2008). Researchers have identified that conformational changes in
antithrombin molecule induced by -asparaginase are responsible for the loss of its activity and formation of
protein aggregates accumulated in ER cisterns (Hernandez-Espinosa et al., 2006). 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 (Mitchell et al., 1994, Miniero et al., 1986). -Asparaginase treatment can also lead to protein C and S
deficiency, which ultimately increases thrombin level and increase the risk of bleeding and thrombosis (Nowak-
Gottl et al., 2001, Athale and Chan, 2003). Generally, 1565% of patients also show hypofibrinogenemia after
asparaginase therapy.

4.3. Pancreatitis, hyperglycaemia, Jump to Section Go


hepatotoxicity
Although, the reason behind such adverse reaction is not well defined but it has been reported to occur due to
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
(Bodmer et al., 2006, Fromenty and Pessayre, 1995, Fromenty and Pessayre, 1997). Patients with liver

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abnormalities through asparaginase treatment rarely show its histological presentation but macro- and
microvesicular liver steatosis have been described (Bodmer et al., 2006, Sahoo and Hart, 2003).

Furthermore, -asparaginase treatment can also increase pancreatic amylase and lipase. Synthesis inhibition of
these enzymes is regulated by -asparagine. In the absence of -asparagine, these enzymes create severe
complications in pancreas (Piatkowska-Jakubas et al., 2008, Raja et al., 2012). -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 -
asparaginase therapy. It has been proposed that a reduction in level of -asparagine and -glutamine in cerebral
tissues is responsible for these adverse reactions. Symptoms of -asparaginase associated CNS related adverse
events include hallucination, drowsiness, and amentia (Chabner and Loo, 1996). It has also reported that -
asparaginase therapy is associated with posterior reversible encephalopathy syndrome (PRES) (Thachil, 2012).

4.4. Resistance to -asparaginase Jump to Section Go


The mechanisms behind the failure and resistance to -asparaginase treatment is still doubtful (Iwamoto et al.,
2007, Williams, 2007). Activity of -asparaginase is dependent on the development of anti- -asparaginase
antibodies, leading to failure of asparagine depletion after re-administration of -asparaginase, and causes -
asparaginase resistance (Panosyan et al., 2004, Douer et al., 2007). These anti- -asparaginase antibodies
neutralize the -asparaginase activity, resulting in more rapid plasma clearance and shorter duration of drug
effect. Antibody development may occur in patients having clinical allergy symptoms (Avramis and Panosyan,
2005, Panosyan et al., 2004, Boos et al., 1996).

Resistance to -asparaginase therapy may also be assumed because of the high level of -asparaginase
synthetase cellular expression, that allows the production of asparagine and hence protein synthesis. Expression
of cellular -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 -asparaginase (Iwamoto et al., 2007, Kaspers et al.,
1995). 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 -asparagine in TEL/AML1 (Stams et al., 2005). 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 (Iwamoto et al., 2007).

Resistance to -asparaginase also arise from the increased expression of asparagine synthetase stimulated by
the tumour cells after exposure to -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 (Iwamoto et al., 2007, Williams, 2007). Co-culture with mesenchymal cells protected
ALL cells from the cytotoxicity caused by -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 (Iwamoto et al., 2007). 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 (Williams, 2007). Other mechanisms
regulating sensitivity of leukemic cells to -asparaginase are still doubtful and unstated. -Asparaginases are
p53-dependent therapeutic agents that cause cytotoxicity by the mechanism of apoptosis. Other key apoptotic
genes associated with -asparaginase resistance are BCL2L13, Harakiri and TNF (Fu and Sakamoto, 2007,
Holleman et al., 2006).

Jump to Section Go
5. Advancement in -
asparaginase discovery
from alternative sources
In order to overcome the limitations of current asparaginase formulations, modified preparations of -
asparaginase have been proposed (Thomas et al., 2010). Occurrence of -asparaginases in fungi, yeasts,
bacteria and animal cells, and their antitumour effects were reviewed in many articles (Adamson and Fabro,
1968, Wriston and Yellin, 1973), but the majority of -asparaginases cannot be translated for human applications.
Despite this, discovery of -asparaginase in several organisms raises optimism about the development of this
therapeutic agent with less adverse reactions.

5.1. Algal asparaginase Jump to Section Go


-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 1.34104M
respectively. The purified enzyme was stable at room temperature for 24 days in sterile solution and showed the
greatest activity at 55C. The Chlamydomonas -asparaginase possess little ability to inhibit the growth of the
Gardner's lymphosarcoma in C3H mice (Paul, 1982). 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 (Holcenberg and
Teller, 1976). Although, the Chlamydomonas asparaginase has satisfied many of these requirements, it Km is
higher than asparaginases employed in chemotherapy (Wriston and Yellin, 1973). Kinetic studies with this
enzyme indicate that the relatively high Km value may have limited its antitumour activity, although other factors
such as serum survival were not investigated (Broome, 1968).

5.2. Plants asparaginase Jump to Section Go

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In comparison to the bacterial enzymes, the plant enzymes have been studied less thoroughly. In plants, -
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 (Lough et al., 1992).

There are two groups of such proteins, called potassium-dependent and potassium-independent asparaginases
(Sodek, 1980). 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 (Lough et al.,
1992).

Withania somnifera L. has been traditionally used in various folklores as a sedative and hypnotic. Recently, in
vitro cytotoxicity of -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 -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 -asparaginase with antitumour activity. W.
somnifera enzyme has less toxicity compared to bacterial -asparaginase (Oza et al., 2010).

5.3. Fungal asparaginase Jump to Section Go


A long time ago, researchers found the -asparaginase activity in Penicillium camemberti (Dox, 1909). In 1930, -
asparaginase of Aspergillus niger was studied by Schmalfuss and Mothes (1930)). Carta de-Angeli et al. (1970))
reported -asparaginase isolated from A. terreus, which suppressed the growth of Walker 256 ascites sarcoma in
rats. Scheetz et al. (1971)) studied the properties of -asparaginase purified from mycelia of Fusarium tricinctum,
and pointed out the inability of the enzyme to suppress the growth of GC3HED lymphosarcoma in mice. Arima et
al. (1972)) examined the extracellular formation of -asparaginase by a variety of micro-organisms and indicated
that the enzyme was produced by several species of Penicillium, Aspergillus, and Nectria. -Asparaginase from
Cladosporium cladosporiodies shows anti-tumour activity against Erlich's ascites in mice (Ali et al., 1993).

Recently, Shrivastava et al. (2010)) found several fungi with -asparaginase activity. Few of them show the
cytotoxic effect against various human cancer cell lines (Shrivastava et al., 2010, Shrivastava et al., 2012). -
Asparaginase 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 (Loureiro et al., 2012).

5.4. Actinomycetes asparaginase Jump to Section Go


-Asparaginase with antineoplastic activity is also found in actinomycetes. Partially purified enzyme with
molecular weight 140Kd, optimum stability at temperature 60C and pH 8.0. -Asparaginase showed a cytostatic
effect in 24h, whereas the cytotoxic effect in 48h against JURKAT and K562 human cancer cell line (Dhevagi
and Poorani, 2006).

5.5. Entrapment of - Jump to Section Go


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. -Asparaginase can enter in the erythrocytes
by reversible osmotic lysis. 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 -asparaginase from anti- -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
half-life 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 (Godfrin and Bertand, 2006).

In addition to the above mentioned alternative sources, human glycosylasparaginase is also investigated due to
their potential of -asparagine hydrolysis into -aspartate and ammonia similar to bacterial asparaginases without
any -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 (Kelo et al., 2009).

Jump to Section Go
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 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 -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 -asparaginase which can pass
all limitations posed by current asparaginase formulations.

Jump to Section Go
Conflict of interest
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9/8/2017 Recent developments in l-asparaginase discovery and its potential as anticancer agent - Critical Reviews in Oncology / Hematology
Authors do not have any personal or financial conflict of interest related to this work.

Jump to Section Go
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.

Jump to Section Go
Acknowledgement
The authors are grateful to Deanship of Scientific Research and Research Center, College of Pharmacy, King
Saud University, Riyadh, Saudi Arabia

Jump to Section Go
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Biography
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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