ONCH_1928 - Recent Developments in L-Asparaginase Discovery and Its Potential as Anticancer Agent

Please citeagent. Crit

ARTICLE IN PRESSONCH-1928; No. of Pages 12

Critical Reviews in Oncology/Hematology xxx (2015) xxxxxx

Recent developments in l-asparaginase discovery and its potential asanticancer 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, Indiab Department of Pharmaceutics, College of Pharmacy, PO Box 2457, King Saud University, Riyadh 11451, Saudi Arabia

c College of Allied Health Professionals, Directorate of Medical Sciences, Government College University, Faisalabad, PakistanDepartment of Microbiology, Vikram University, Ujjain, MP, India

Contents

1. Introdu2. Mechan3. Curren

3.1. C

4. Problem4.1. H4.2. C4.3. P4.4. R

5. Advanc5.1. A5.2. P5.3. F5.4. A5.5. E

6. ConcluConflicReviewAcknowReferenBiograp

Abstractl-Asparag

cancers from

CorresponTel.: +966 54

E-mail ad1 These aut

http://dx.doi.o1040-8428/ this article in press as: Shrivastava A, et al. Recent developments in l-asparaginase discovery and its potential as anticancer Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.01.002

e Department of Biological Sciences, Rani Durgavati University, Jabalpur, MP, IndiaAccepted 5 January 2015

ction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00ism of l-asparaginase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

t asparaginase formulations: a comparative evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00urrent asparaginase formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3.1.1. Native E. coli asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.1.2. Erwinia asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.1.3. PEG-asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

s associated with clinical application of bacterial asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00ypersensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00oagulation disorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00ancreatitis, hyperglycaemia, hepatotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00esistance to l-asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

ement in l-asparaginase discovery from alternative sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00lgal asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00lants asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00ungal asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00ctinomycetes asparaginase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00ntrapment of l-asparaginase in erythrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

sion and future direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00t of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00ers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00ledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00ces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00hies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

inase (EC3.5.1.1) is an enzyme, which is used for treatment of acute lymphoblastic leukaemia (ALL) and other related blood a long time. This enzyme selectively hydrolyzes the extracellular amino acid l-asparagine into l-aspartate and ammonia, leading to

ding author at: Department of Pharmaceutics, College of Pharmacy, PO Box 2457, King Saud University, Riyadh 11451, Saudi Arabia.2854355.dress: [email protected] (A.A. Khan).hors have equal contribution.

rg/10.1016/j.critrevonc.2015.01.002 2015 Elsevier Ireland Ltd. All rights reserved.d

Please cite this article in press as: Shrivastava A, et al. Recent developments in l-asparaginase discovery and its potential as anticanceragent. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.01.002

ARTICLE IN PRESSONCH-1928; No. of Pages 122 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 asparaginasesare used for treatment purpose but offers scepticism due to a number of toxicities, including thrombosis, pancreatitis, hyperglycemia, andhepatotoxicity. Resistance towards bacterial asparaginase is another major disadvantage during cancer management. This situation attractedattention of researchers towards alternative sources of l-asparaginase, including plantl-asparagina ects realso provide fromalternative to 2015 Else

Keywords: l- Erwina

1. Introdu

l-Asparfor manageand other Kidd identtrol the protransplanteand repeateserum wer

sion of lymmice grew days [1]. Manti-lymphthat guinea[2]. In the phoma cellamino acidlation rapidunder l-asplost guineamice, whileity even aftto guinea pwhile otherto complemasparaginaantilymphoother reseathis enzymhas anti-lylike horse sparable actagainst maagainst only some cancer but not all. Guinea pig serum wasable to inhibit certain lymphoma cell lines in vivo, but thesame activity was not possible with in vitro assays, providedthat guinea pig serum is also a rich source of complementand proposed anticancer activity against cell lines may bedue to complement mediated cytotoxicity. l-Asparaginase isan inhibitor of complement in whole guinea pig serum dur-ing in vitro assay [4]. Broome performed research to find outthe relative contribution of complement and l-asparaginase

ticancjor antal triawy [6ility fmmo

anagetime, ion. Tme wifor rearagintion, pe prosn as a

echan

linicale as a

a mo

allogrnit is . coli e trea

ren thnsion ther o

acutehood eoplasool oits the

G1-phase and ultimately apoptosis in susceptible leukemiccells [9,10]. Unlike normal cells, leukemic cells and othercancer cells have little or no asparagine synthetase and hencethey are not able to carry out the de novo asparagine synthe-sis, resulting in inhibition of protein synthesis and subsequentdeath of the tumour cells [11] (Fig. 1).

Normal cells are protected from asparagine requirementdue to their ability to produce this amino acid [12]. Unlikeconventional cancer therapy, l-asparaginase therapy is highlyse as an anticancer agent, its mechanism of action, and adverse effs an outlook for recent developments in l-asparaginase discovery

current formulations.vier Ireland Ltd. All rights reserved.

Asparaginase; Acute lymphoblastic leukaemia; Anticancer; l-Asparagine;

ction

aginase is an important chemotherapeutic agentment of acute lymphoblastic leukaemia (ALL)

hematopoietic malignancies. In the early fifties,ified that guinea pig serum has the ability to con-gression of murine lymphoma. The mice were

d with lymphoma cells during this experimentd intraperitoneal injections of normal guinea pig

e given to them. This treatment led to a regres-phoma and survival of treated mice while controllymphoma progressively and died within 2030uch earlier the discovery of guinea pig serum

oma activity, it was observed by Clementi (1922), pig serum is also a rich source of l-asparaginaseearly sixties, guinea pig serum susceptible lym-s were grown in cell culture media devoid of the

l-asparagine, this inadequacy declined cell popu-ly, but some cells survived and began to proliferateargine limitation. These cells were found to have

pig serum susceptibility after transplantation in original lymphoma cells retained this susceptibil-er transplantation in mice. The lost susceptibilityig serum was restricted to l-asparagine limitation,

amino acids, purines and pyrimidines were unableent this effect. Therefore, it was concluded that l-

se present in guinea pig serum is responsible for itsma activity [3]. These observations also attractedrchers for the detection of anticancer potential ofe, and it was found that the only guinea pig serummphoma activity, while sera from other animals,erum and rabbit serum were not able to show com-ivity [1]. Furthermore, the same sera was testedny cancer types and observed that it is effective

in ana ma

clinicDoloits aband afor mlong reactenzygoal l-aspof acfuturcatio

2. M

Cactivwith crystsubuas Efor thchildexpaand ohoodchildantining pinhibs and fungi. Present article discusses about potential oflated to current asparaginase formulations. This article

alternative sources and their potential as a less toxic

se; Kidrolase; PEG-asparaginase

er activity, and concluded that l-asparaginase isicancer agent in guinea pig serum [3,5]. The firstl on l-asparaginase was performed in 1966 by]. It is a major chemotherapeutic agent due toor hydrolysis of l-asparagine into l-aspartic acidnia. Current asparaginase formulations are usedment of acute lymphoblastic leukaemia from abut these formulations are not free from adverseherefore the search for alternative sources of thisth reduced or no adverse reaction is an importantsearchers. This article discusses the potential ofase enzyme as anticancer agents, its mechanismroblems associated with bacterial asparaginases,pects in l-asparaginase discovery and their appli-less toxic alternative.

ism of l-asparaginase activity

ly used l-asparaginase enzyme from E. coli is tetrameric protein with identical subunits, eachlecular weight of 35.6 kDa according to x-rayaphic data [7]. The molecular formula for eachC1377H2208N382O442S17. Chemically it is knownl-asparagine amidohydrolase. It is widely usedtment of haemopoietic diseases such as ALL inat results from the monoclonal proliferation andof lymphoid blasts in the bone marrows, blood,rgans. ALL correspond to the most common child-

leukaemia contributing to approximately 80% ofleukaemias and 20% of adult leukaemias [8]. Thetic activity results from depletion of the circulat-

f l-asparagine by l-asparaginase, which in turn protein synthesis, causes cell cycle arrest in the


ARTICLE IN PRESSONCH-1928; No. of Pages 12A. Shrivastava et al. / Critical Reviews in Oncology/Hematology xxx (2015) xxxxxx 3

discriminatas a remedplasma cleaquent injecand a numimmune resbe serious

The abobe due to sel-Glutamintion of plais enzymaticantly lowAs glutamil-asparaginasparagineof l-asparasparaginetic effect o

l-asparagin this article in press as: Shrivastava A, et al. Recent developments in l-a Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.0

Fig. 1. General mechanism behind selective toxicity

ory. The main restrictions in using l-asparaginaseial agent is its premature inactivation, more rapidrance and shorter duration of drug effect, thus fre-tions are required to maintain a therapeutic level,ber of side effects like allergies, development ofponses and finally anaphylactic shock, that might

and life-threatening [1315].ve-mentioned side effects of l-asparaginase mayveral reasons, including its l-glutaminase activity.ase activity of enzyme results in some reduc-sma l-glutamine level [1618]. l-Asparaginaseically active against glutamine, but with a signif-er affinity against glutamine than l-asparagine.ne acts as an amino group donor for the enzymee synthetase for de novo biosynthesis of l-

, therefore, the decreased glutamine level becauseaginase exposure also help in sustaining the

level reduction and thus contributes the therapeu-f l-asparaginase [19] which can be explained as:

el-asparaginase l-aspartic acid + NH3

l-glutamin

De novo

l-aspartic aAsparagine

Leukaem

Aspara No P

The exanot clear, to proceed(Fig. 2). Hocontributesis also relacer treatmesparaginase discovery and its potential as anticancer1.002

of l-asparaginase.

el-asparaginase l-glutamic acid + NH3 biosynthesis:

cid + NH3 (From glutamine) Synthetase

l-asparagine

ia cell after l-asparaginase:gine No l-asparaginerotein Synthesis Cell Death

ct mechanism of l-asparaginase action is stillalthough hydrolysis of l-asparagine is known

in two steps via a -acyl-enzyme intermediatewever, the l-glutaminase activity of asparaginases

to the associated side effects, but this activityted with cell growth inhibition in certain can-nts [20,21]. l-Asparaginase induced reduction of



Fig. 2. Struct raginase reaction, covalent intermediate (-Acyl-Enzyme) is formedthrough nucle his intermediate later convert into l-aspartate and gives final reactionproducts as l-

asparagineway and itan intracel(known as of downstrlation of thor p70s6k)tein 1 (4Eproteins atthat inhibitaffect leukactivity. Inhas been fprocess thstructurallymTORC1 ivates proteacid abundteins Agt1/induced redlular signacells.

3. Currena compara

ALL antively treaanti-leukemfollowing of the aspserum cleaof asparagiopment of system [25either fromliver or the ble formuladespite its ALL.

Curre

Asparlso innia, Setobac

spp. ellus suaraginrate h

of l-ass an

ns avaanthe

the idely

(ElspegylatJ, UwiniaUK). line trormers are

wa Hrkusence) Paural illustration of the of l-asparaginase reaction mechanism. During l-aspaophilic attack by the enzyme. Green arrows indicate a nucleophilic attack. Taspratate and ammonia.

and glutamine level is linked with mTOR path-s subsequent inhibition. This pathway includeslular molecule mammalian target of rapamycinmTOR). Inhibition of mTOR leads to inhibitioneam events in its pathway including phosphory-e protein serine threonine kinase (p70S6 kinase

and eukaryotic initiation factor 4E-binding pro--BP1) which suppresses synthesis of ribosomal

mRNA translation level [22]. It has been foundion of mTOR pathway by l-asparaginase greatlyaemia cells and contributes to its antileukaemic

a separate study on acute myeloid leukaemia, itound that l-asparaginase induces an autophagicrough inhibition of mTORC1 (mTOR has two

distinct complexes mTORC1 and mTORC2).s an important regulator of cell growth which acti-in translation and inhibit autophagy under aminoance by regulating the autophagy related pro-ULK and ATG13 [23]. Therefore l-asparaginaseuction of amino acid level is associated with cel-

lling which in turn leads to death of leukeamic

t asparaginase formulations:tive evaluation

d other lymphoid malignancies have been effec-

3.1.

l-but aErwiAcinriumBacil-aspmodetion posseratiochrys

Inare w

nase

its Pter, Nof ErLtd, ond the flation(KyoLeveFran this article in press as: Shrivastava A, et al. Recent developments in l-a Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.0

ted with asparaginase for many years. Theic activity of asparaginases depends upon the

factors. (i) The rate of hydrolysis, and the Kmaraginase. (ii) The pharmacological factors ofrance of the enzyme [24]. (iii) The developmentnase resistance in tumour cells [18]. (iv) Devel-anti-asparaginase antibodies by the host immune]. (v) The increased contribution of asparagine

de novo biosynthesis of asparagine within theinput from the nutrient intake [26]. The best possi-tion and dosage of asparaginases is still disputed,use as a vital drug in all treatment protocols for

Table 1Clinical pharm

Formulation

Native E. coliasparaginasePEG-asparagi

Erwinia asparnt asparaginase formulations

aginases have been found not only in mammals, birds, plants, bacteria (like Escherichia coli,almonella, Mycobacteria, Pseudomonas, andter species etc.), and fungi (Aspergillus, Fusa-tc.) [27]. Bacillus licheniformis, a member of thebtilis group has recently assessed for production ofase with low glutaminase activity [28]. Similarly,alophilic bacteria has recently showed produc-

sparaginase [29]. Although not all asparaginasesti-cancer activity and currently, the only prepa-ilable for medical use are the E. coli, Erwiniami asparaginases, and their derivatives (Table 1).United States, three asparaginase formulations

used against ALL: native E. coli asparagi-ar; Merck & Co., Inc., West Point, PA, USA),ed form (Oncaspar; Enzon, Inc., Bridgewa-SA) and Erwinase, the product from cultures

chrysanthemi (Ipsen-Speywood PharmaceuticalsThe latter formulation is approved in UK as sec-eatment for patients having hypersensitivity to

two forms. These native asparaginase formu- offered under different brand names; Medacakko, Kogyo, Japan), Crasnitin (Bayer AG,, Germany), Leunase (Sanofi-Aventis, Paris,

ronal, and Kidrolase etc. in Europe and Asia.sparaginase discovery and its potential as anticancer1.002

acology of asparaginases with frequently administered doses.

Elimination half life Dosage

2630 h 6000 IU/m2 threetimes/week

nase 5.57 days 20002500 IU/m2every 2 or 4 weeks

aginase 16 h 6000 IU/m2daily ten doses, thenthree doses weekly, or30000 IU/m2daily ten doses ininduction

saadHighlight

saadNoteErwinaze

saadHighlight

saadHighlight

saadNote(Jazz Pharmaceuticals, USA)

saadNoteThis asparaginase from Erwinia is registered in UK and other European countries as Erwinase.



Table 2Asparaginase formulations therapy in naive adult patients.

Region

North AmericAustralia, NZealand

Europe

Other countri

However soreferred in

3.1.1. NatiThe asp

for ALL fearly 1990was not lonMedac wnon-Hodgkusing the siMedac lelopathies li

In compferences wwere produhigher biothe dose tto achievepharmacokThe formeasparagineplete asparof the childdisparities particularlyneed to adjthe dosing number of the studiescarried out apeutic ranfrom studiand CSF wfrom the co2500 IU/mtoxicity as[31].

3.1.2. ErwErwinia

patients havlicensed fomany coun

E. coli preparations with reference to immunogenicity andless induction of coagulation disorders [36].

e efficacy of Erwinase has been doubted as the activ-as sig

of 1 to 94over ceen fosage

[37, asparaly 26%found on-peased f.

furthepy is aginanistratadmina leve

ess obgh IM

On thean en

lete awinia

[37].througtion w

formant disnistrat

ordents, otse of dular p

and ly basnding nia asover ts furth

. PEGowadaa halFirst Line Second Line

a, UK,ew

E. coli-asparaginaseor PEG-asparaginase

Erwinia asparaginaseor PEG-asparaginase

E. coli-asparaginaseor PEG-asparaginase

Erwinia asparaginase

es E. coli-asparaginase Erwinia asparaginaseor PEG-asparaginase

me of these are no longer available now and onlythe literature [24,30] (Table 2).

ve E. coli asparaginasearaginase has improved event-free survival (EFS)rom 80% in the past few years. Ins, Crasnitin (Bayer AG, Leverkusen, Germany)ger available. Therefore asparaginase formulationas included in Berlin, Frankfurt, Muenster-ALL-ins lymphoma (BFM-ALL-NHL) protocols,milar treatment regimen. However treatment withads to more adverse reactions, especially coagu-ke haemorrhagic and thrombotic events.arison of Medac versus Crasnitin, significant dif-ere described despite the fact that both enzymescts of E. coli strains. Medac showed significantlylogical activity so it was possible to decreaseo 2550% of the dosage of Crasnitin in order

sufficient asparagine depletion under carefulinetic and pharmacodynamic monitoring [31,32].r was more effective than crasnitin, not only in

depletion but also in glutamine depletion. Com-agine depletion was attained in more than 90%ren in treatment with Medac asparaginase. Largeexist between the various products and half life is

reliant on the preparation. Although there is noust the individual dose administered, alteration ofinterval is compulsory, resulting in variation to thedosages [33,34]. In a limited number of patients,

with reduced doses of asparaginase Medac werebecause asparaginase activity higher than the ther-ge may lead to increased toxicity. This is apparentes that complete asparagine depletion in serumas accomplished by reducing the induction dosemmon 10000 IU/m2 to 5000 IU/m2 and even to

2 administered at 3-day intervals in order to reduce

Thity wlevelparedMorehas blent d92.6%pletein onwas

for nincretaken

Atheraasparadmi(IV) plasmwas lthrou[39].in mcompof Erphasemen

depleto thenificaadmi

Inpatiein caparticcases

ativedepeErwiMoresitate

3.1.3N

plasm this article in press as: Shrivastava A, et al. Recent developments in l-a Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.0

sociated with highly active Medac asparaginase

inia asparaginase asparaginase (Erwinase) has been used to treating allergy to E. coli asparaginases. Erwinase was

r use in patients in Europe and is now available intries [35]. Erwinia asparaginase is superior to the

genicity ofwith polyeas PEGylaedition of tion of E. glycol (PEations in c[33,45,46].sparaginase discovery and its potential as anticancer

nificantly lower after the first exposure: a trough00 U/L was achieved in 33% of patients as com-.5% of patients treated with E. coli asparaginase.onsiderably lower i.e. 26% asparagine depletion

ound after a second exposure despite of equiva-s in contrast to the patients in E. coli group i.e.38]. In some similar studies with Erwinase com-gine depletion was achieved on day 3 of treatment

of patients, whereas in some patients no depletion[32]. Therefore, using Erwinia as replacements

gylated E. coli asparaginases, a higher dose andrequency of treatment is the foremost step to be

r necessary feature in the Erwinia asparaginasethe route of administration. A slower increase ofse activity is observed after intramuscular (IM)ion due to the depot effect while intravenousistration results in concomitant elevated peakls. Asparaginase activities below the desired levelserved after Erwinia asparaginase administration

route as compared to intravenous administratione other hand, no significant differences were foundzyme activity or frequency of samples showingsparagine depletion after IV or IM administration

asparaginase administered during the induction Likewise, in patients receiving more intense regi-h IM or IV route, analogous complete asparagineas found in the induction phase [40]. With regardstion of asparaginase neutralizing antibody, no sig-similarities were found among the both routes ofion as a re-induction regimen [41].r to ensure maximum survival benefit in ALLher asparaginase preparation should be employedevelopment of anti-asparaginase antibodies to areparation. The assessment of silent inactivation

the degree of serum asparagine depletion is rel-ed on asparaginase level investigation. In brief,upon regulatory factors, practice and availability,paraginase is a valid second or third-line therapy.he optimized use of Erwinia asparaginase neces-er clinical and pharmacokinetic studies [30].

-asparaginaseys, the most appropriate approach to enhance thef-life and trim down the immunogenicity and anti-

many therapeutic agents is its covalent couplingthylene-glycol (PEG) and the process is knowntion [4244]. PEG-asparaginase is the modifiedl-asparaginase formed by the covalent conjuga-coli asparaginase to monomethoxypolyethyleneG) resulted in significant pharmacokinetic vari-omparison with the native E. coli formulations

PEGylation of proteins and enzymes results in1.002

saadHighlight

saadNote(Erwinaze, USA, Erwinase UK)



aive a

covering ofsize and ensimilar chei.e. reactionand optimuPEG-asparthe E. coli PEG-aspardisparities l-asparaginmanufacturwhile the pasparagina

The elitimes longtimes longrepresents decreasingapeutic effiformulationEurope as aallergic to nabsorption asparagination half-liin plasma asparagina

The roumaximum levels are atration [18]and glutamasparaginecytotoxic e

of ause of

patienot ons, but e dosan be

ite thethe ce-asparaginef aspa

dose ens af

IntravFig. 3. Regional PEG-asparaginase therapy in N

few surface sites, thus increasing their molecularhancing steric-hindrance. PEG-asparaginase hasmical properties like native E. coli l-asparaginase

temperature of 50 C, isoelectic point at pH 5.0m activity at pH 7.0 [47]. The currently availableaginase formulation in most of the countries isderived asparaginase, Oncaspar [48]. Within theaginase products from different countries minormay be found. PEG-asparaginase from the E. coliase obtained from Merck, Sharp and Dohme areed by Enzon in US and marketed as Oncaspar,roduct is derived from the Kyowa Hakko native

se protein in Europe [49,50].mination half life of PEG-asparaginase is fiveer than the native E. coli preparations and nine

routebecaALLwill level

Thels cdespinto PEGaspartion osame

happ[53]. this article in press as: Shrivastava A, et al. Recent developments in l-a Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.0

er than that of Erwinia i.e. around six days. Thisan imperative improvement in therapy through

the number of injections needed to achieve ther-cacy in naive patients [19]. In the USA, the

is being approved as first-line therapy whilst in second-line treatment restricted to known patientsative asparaginases (Fig. 3). In the cited study thefrom the injection site was 1.7 days, whereas these activity detectable for a longer period (elimina-fe; 5.5 days) whereas the peak enzymatic activityat 5 days after an intramuscular dose of PEG-se [45].te of administration is the main factor, becauseamino acid depletion and peak enzyme activityttained within 5 days after intramuscular adminis-. This step by step depletion of serum asparagineine may allow increased production of hepatic

synthetase and asparagines, therefore diminishingffects exerted on the cancer cell. The intravenous

sufficient topared to

Please cite in l-aagent. Crit


important and the best possible method should be defined forthe use of l-asparaginase in ALL therapy. The productionof neutralizing anti-asparaginase antibodies may be avoidedby the alte(Native E.asparaginaFurthermorlevels shousilent hypeof asparagi

4. Problembacterial a

Despiteopment, cuMajor advasparaginasubsequentuse of aspa

Allergicantibody itoxicity dutions exagproper imwith steroidreduction asparaginanase in comof hyperseQuinckes oThe inciden(IM) administration. Greactions aasparaginamany reasoing remissiproper immtion and susymptoms during induhypersensithere is rapsensitivity suboptimal

Productfor resistanhigh level tion reducecases [60,6preparationof allergic immune su

that belong to immunologic sensitization to a foreign proteinand another related to the protein synthesis inhibition [62].

Hyper

s disc reac

rash ass, antion o

fromess, iria, rema, aginadysfucidosin synulopathas b

g fromaraginaginaring a

Coagu

sparagd abn

n (inhde an

are alaring.14%bin a

as facth formolyticthat coed by

ity andisternd sideotein inoge

partient c

ately eedingnts alpy.

Pancr

lthougell d this article in press as: Shrivastava A, et al. Recent developments

rnate use of various asparaginase formulations coli asparaginase PEG-asparaginase, Erwiniase) during the different phases of ALL therapy.e, anti-asparaginase antibodies and asparagineld be monitored strictly to facilitate detection ofrsensitivity. In that case, the change in formulationnase may be able to improve its activity [45].

s associated with clinical application ofsparaginase

significant advancement in l-asparaginase devel-rrent formulations are not free from limitations.erse events are related to immune reaction tose protein. Furthermore asparagine depletion and

protein synthesis inhibition also aid in restrictingraginase.

reactions are primarily due to anti-asparaginasen circulation and lead to major reason fore to l-asparaginase [18,25]. Such complica-gerate when asparaginase was given withoutmune-suppressive therapy. Immunosuppression

reduces the chances of hypersensitivity throughof high titre antibodies. Production of antise antibody was higher with native asparagi-

paring to PEG-asparaginase. Clinical symptomsnsitivity include anaphylaxis, pain, oedema,edema, urticaria, erythema, rash and pruritis [25].ce of skin reactions was high with intramuscularistration compared with intravenous (IV) admin-enerally, it has been observed that hypersensitivityre found during post-induction phase when l-se is not provided for week or months. There arens for the low frequency of allergic reactions dur-on induction, like there is a delay for generation ofune response due to delay in complement activa-bsequent production of antibodies [26]. Allergicmight hide by intensive corticosteroids therapyction phase [26]. About 30% patients show silent

tivity or silent inactivation. During this conditionid inactivation of asparaginase instead of hyper-reaction which leads to asparagine depletion at a

level [25,58,59].ion of anti-asparaginase antibody is responsiblece in asparaginase therapy and also causes the

of asparagines in blood plasma [50]. This condi-s the therapeutic efficacy of asparaginase in some1]. All these conditions demand the alternative

of asaparaginase for removal or low frequencyreactions [59]. The toxicities of l-asparaginaseppression may divide into two types; firstly those

4.1.

Alogicand distresentarangesicknurticaerythasparliver ketoaprotecoag

It ferinl-aspasparrequi

4.2.

Arelateserpiinclutionscompand 1thromwell whicprotefied inducactivER cciateon prplasmvatedtreatmultimof blpatiethera

4.3.

Anot w Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.0

sparaginase discovery and its potential as anticancer

sensitivity

ussed earlier, bacterial proteins induce immuno-tions leading to localized, transient erythemat the site of injection to urticaria, respiratoryd acute life-threatening anaphylaxis. Clinical pre-f asparaginase hypersensitivity reactions may, allergic reactions, anaphylaxis (rare), serumtching and swelling of extremities, oedema,ash and broncospasm, localized or generalizedand other clinically related reactions. Moreover,ses treatment can also lead to organ toxicities,nction, pancreatitis and related hyperglycemia,is, glucosuria, cerebral dysfunction, decreasedthesis, hypofibrinonemia, hypercoagulable state-hies, hypoalbuminemia [26].een observed that about 60% patients were suf-

hypersensitivity reactions during therapy ofase from E. coli. Even E. coli derived PEG

se also shows hypersensitivity reactions, thus shift to another form of asparaginase [63].

lation disorder

inase therapy can also lead to several coagulationormalities due to severe acquired deficiency ofibitor of serine protease) proteins. These proteinstithrombin and 1 antitrypsin. Such complica-so higher with native forms of l-asparaginase in

to PEG asparaginase with the risk of 2.115% respectively [55,64]. Physiological inhibition of

nd other coagulation factor like IXa, Xa, XIa asor VII is majorly regulated through antithrombin,s an irreversible link with enzyme leading to its

activity inhibition [65]. Researchers have identi-nformational changes in antithrombin molecule

l-asparaginase are responsible for the loss of its formation of protein aggregates accumulated in

s [66]. Furthermore, coagulation disorders asso- effects are produced due to the effect of drugsynthesis, reduction in antithrombin, fibrinogen,n, and factors IX and X with prolongation of acti-al thromboplastin time [67,68]. l-Asparaginasean also lead to protein C and S deficiency, whichincreases thrombin level and increase the risk

and thrombosis [69,70]. Generally, 1565% ofso show hypofibrinogenemia after asparaginase

eatitis, hyperglycaemia, hepatotoxicity

h, the reason behind such adverse reaction isefined but it has been reported to occur due to1.002



defects in protein synthesis. Asparaginase treatment asso-ciated liver toxicity includes oxidative stress, glutaminedeficiency, decreased hepatic protein synthesis, and lateron impairmPatients wiment rarelymicrovesic

Furthermpancreatic enzymes isasparaginepancreas [diabetes inthrough affexocrine (d

In addsymptoms asparaginain level ofsues is resof l-asparinclude halalso reportposterior re

4.4. Resist

The measparaginal-asparaginl-asparagindepletion causes l-l-asparaginactivity, resduration ofpatients ha

Resistanbecause of lar expresshence protesynthetase and the hytheir increaversely, inover expreinated and in TEL/AMthat cellulashown to bALL with thomolog lefusion gene

Resistanexpressioncells after e

of asparagine synthetase by the mesenchymal cells of thebone marrow which constitute the marrow microenvironmentfor leukaemia cells where the leukemic cells mature [78,79].

ultureytotoxeffect

regults of metase

ested marro

entratient [7tical in

and inimaanismagina53-depe meciated kiri an

dvancnative

orderformu

been i, yeas

effecrity ofcationrganispeutic

Algal

Aspared tomined

m we

purifieays int 55 C

abilitarcomnti-tumysiolome by

rate o87]. Aed maginaith th this article in press as: Shrivastava A, et al. Recent developments

ent of beta-oxidation in mitochondria [7173].th liver abnormalities through asparaginase treat-

show its histological presentation but macro- andular liver steatosis have been described [71,74].

ore, l-asparaginase treatment can also increaseamylase and lipase. Synthesis inhibition of these

regulated by l-asparagine. In the absence of l-, these enzymes create severe complications in65,75]. l-Asparaginase therapy can also induce

some patients due to decreased insulin synthesisecting both the endocrine (insulin-secreting) andigestive enzyme-secreting) cells of the pancreas.ition to above complications, CNS relatedare another adverse events associated with l-

se therapy. It has been proposed that a reduction l-asparagine and l-glutamine in cerebral tis-ponsible for these adverse reactions. Symptomsaginase associated CNS related adverse eventslucination, drowsiness, and amentia [76]. It hased that l-asparaginase therapy is associated withversible encephalopathy syndrome (PRES) [77].

ance to l-asparaginase

chanisms behind the failure and resistance to l-se treatment is still doubtful [78,79]. Activity ofase is dependent on the development of anti-ase antibodies, leading to failure of asparagine

after re-administration of l-asparaginase, andasparaginase resistance [25,51]. These anti-ase antibodies neutralize the l-asparaginaseulting in more rapid plasma clearance and shorter

drug effect. Antibody development may occur inving clinical allergy symptoms [24,25,32].ce to l-asparaginase therapy may also be assumedthe high level of l-asparaginase synthetase cellu-ion, that allows the production of asparagine andin synthesis. Expression of cellular l-asparagineis found low in B-lineage ALL with TEL/AML1per-diploid subtype and may be an indication ofsed sensitivity to l-asparaginase [78,80]. Con-

some new studies of TEL/AML1 ALL cells,ssion of asparagine synthetase mRNA was orig-did not correlate with resistance to l-asparagineL1 [81]. In other words, it can be summarizedr asparaginase synthetase over expression was

e higher in T-lineage ALL and lower in B-lineageranslocation-erythroblastosis virus E26 oncogeneukaemia-acute myeloid leukaemia 1 TEL-AML1

[78].ce to l-asparaginase also arise from the increased

of asparagine synthetase stimulated by the tumourxposure to l-asparaginase and from the expression

Co-cthe cding downeffecsynthsuggboneconc

ronm

is cricellsof mmechasparare pby thassoc

Hara

5. Aalter

Innase

havefungmour

majoapplieral othera

5.1.

l-purifideterand KThe 24 dity alittlephosfor aat phenzyslowKm [satisfiasparies w Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.0


with mesenchymal cells protected ALL cells fromicity caused by l-asparaginase, and this shiel-

correlated with asparagine synthetase levels, i.e.ation by RNA interference decreased the shieldingesenchymal cells, whereas enforced asparagine

expression give enhanced protection. The authorsthat mesenchymal cells-mediated niches in thew form a safe haven for ALL blasts by increasingons of asparagine in the leukemic cell microenvi-8]. It is yet to be decided whether this association

vivo or if the relationship between the leukemicmesenchymal cells contributes to developmentl residual disease in ALL patients [79]. Others regulating sensitivity of leukemic cells to l-

se are still doubtful and unstated. l-Asparaginasesendent therapeutic agents that cause cytotoxicityhanism of apoptosis. Other key apoptotic geneswith l-asparaginase resistance are BCL2L13,d TNF [45,82].

ement in l-asparaginase discovery from sources

to overcome the limitations of current asparagi-lations, modified preparations of l-asparaginaseproposed [83]. Occurrence of l-asparaginases ints, bacteria and animal cells, and their antitu-

ts were reviewed in many articles [84,85], but the l-asparaginases cannot be translated for humans. Despite this, discovery of l-asparaginase in sev-ms raises optimism about the development of this

agent with less adverse reactions.

asparaginase

aginase from a Chlamydomonas species has been near homogeneity (78 units/mg of protein) as

by disc-gel electrophoresis. Its molecular weightre about 275Kd and 1.34 104 M respectively.d enzyme was stable at room temperature for

sterile solution and showed the greatest activ-. The Chlamydomonas l-asparaginase possess

y to inhibit the growth of the Gardners lym-a in C3H mice [86]. As an important requirement

our activity, this enzyme showed good activitygical pH and temperature, no inhibition of the

reaction end products, no cofactor requirement,f clearance from the serum and relatively lowlthough, the Chlamydomonas asparaginase has

any of these requirements, it Km is higher thanses employed in chemotherapy [85]. Kinetic stud-is enzyme indicate that the relatively high Km1.002



value may have limited its antitumour activity, although otherfactors such as serum survival were not investigated [88].

5.2. Plants

In comphave been the major nare utilizedsues. Aspaof nitrogenother legum

There ardependent The plant aany signific23% identiinase [89].

Withanivarious foin vitro cywas observthe fruits oinhibitory [3-(4,5-dimmide] (MTresearch waasparaginahas less tox

5.3. Funga

A long activity inasparagina& Mothesasparaginagrowth of et al. (1971from myceinability oflymphosarcthe extracemicro-orgaby several [96]. l-Asshows anti[97].

Recentlwith l-aspaeffect agaiAsparaginashow no cyanti-prolifeHL60 [100

5.4. Actinomycetes asparaginase

l-Asparaginase with antineoplastic activity is also foundtinomht 140.0. l-eas th

huma

Entra

ythrod byansferum. lsible fficien

e is nrocytespara

neutrasing rpy prend theas a n

degrarom sw fre

atients additn glyctial oonia taminraginabacterity [10

onclu

sparagent o

due tchemo

mild hts of a

to thition,

majoactionl-condvantnzymenic c

has b this article in press as: Shrivastava A, et al. Recent developments

asparaginase

arison to the bacterial enzymes, the plant enzymesstudied less thoroughly. In plants, l-asparagine isitrogen storage and transport compounds, those

in protein synthesis in the actively growing tis-ragine is the major nitrogen assimilatory product

fixation and nitrate reduction in Lupinus and manyes [89].e two groups of such proteins, called potassium-and potassium-independent asparaginases [90].sparaginase amino acid sequences did not haveant similarity with microbial asparaginase but wascal and 66% similar to a human glycosylasparag-

a somnifera L. has been traditionally used inlklores as a sedative and hypnotic. Recently,totoxicity of l-asparaginase from W. somniferaed in a study where enzyme was purified fromf W. somnifera L. The antitumour and growth

effect of the l-asparaginase was assessed usingethyl-thiazol-2yl)-2,5-diphenyl-tetrazolium bro-T) calorimetric dye reduction method. Thiss proposed as first report of the plant containing l-

se with antitumour activity. W. somnifera enzymeicity compared to bacterial l-asparaginase [91].

l asparaginase

time ago, researchers found the l-asparaginase Penicillium camemberti [92]. In 1930, l-se of Aspergillus niger was studied by Schmalfuss

[93]. De-Angeli et al. (1970) reported l-se isolated from A. terreus, which suppressed theWalker 256 ascites sarcoma in rats [94]. Scheetz) studied the properties of l-asparaginase purifiedlia of Fusarium tricinctum, and pointed out the

the enzyme to suppress the growth of GC3HEDoma in mice [95]. Arima et al. (1972) examined

llular formation of l-asparaginase by a variety ofnisms and indicated that the enzyme was producedspecies of Penicillium, Aspergillus, and Nectriaparaginase from Cladosporium cladosporiodies-tumour activity against Erlichs ascites in mice

y, Shrivastava et al. (2010) found several fungiraginase activity. Few of them show the cytotoxic

nst various human cancer cell lines [98,99]. l-se from Aspergillus niger was also obtained whichtotoxicity against normal human cells, but showrative effect against leukemic cell line RS4; and].

in acweigpH 8wherK562

5.5.

Ertigatebe trmedirever

and eThererythto l-amay

Utheralife acells rapidism fthe lofor p

Inhumapotenamm

l-glulaspawith activ

6. C

Atreatmariseas a

fromeffectionsdeplein thegic reglycoical aThe eantigfore, Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.0


ycetes. Partially purified enzyme with molecularKd, optimum stability at temperature 60 C and

Asparaginase showed a cytostatic effect in 24 h,e cytotoxic effect in 48 h against JURKAT andn cancer cell line [101].

pment of l-asparaginase in erythrocytes

cytes can be used as a microbioreacter. As inves- Ataullakhanov et al. (1985), asparagine canred into human erythrocytes from the external-Asparaginase can enter in the erythrocytes byosmotic lysis [102]. The enzyme remains activet in the erythrocytes during the circulation flow.

o significant alteration of half life of transfuseds. The erythrocyte membrane provides protectionginase from anti-l-asparaginase antibodies whichlize or significantly reduce enzyme activity.ed blood cells as a micro-bioreactor for enzymesents many advantages, especially increased half-

reduction in hypersensitivity. Therefore, using redew entrapped form would protect enzymes from adation in the blood and provide the safety organ-

ide effects. Due to prolonged one month half-life,quency of injections would be more comfortable

with this preparation [103].ion to the above mentioned alternative sources,osylasparaginase is also investigated due to theirf l-asparagine hydrolysis into l-aspartate andsimilar to bacterial asparaginases without anyase activity. It is suggested that human glycosy-ses can be used to reduce side effects associatedial asparaginases due to absence of glutaminase4].

sion and future direction

inase therapy is an important component in thef children with ALL. However complications mayo certain side effects of the enzyme when usedtherapeutic agent. The complications may rangeypersensitivity to anaphylactic shocks. The toxic

sparaginase are related primarily to immune reac-s bacterial protein and to the effects of asparagineand its subsequent inhibition of protein synthesisr glands such as the liver and pancreas. The aller-s are the most prominent toxicities. Polyethylene

jugated asparaginase has significant pharmacolog-ages over native Escherichia coli asparaginase.e derived from Erwinia carotovora does not shareross-reactivity with the E. coli enzyme and, there-een used when patients develop hypersensitivity1.002



to the E. coli enzyme. This condition attracted scientist fordiscovering this activity in many other organisms. In fact theresearch gathers optimism about prospects of l-asparaginaseobtained frmanagemehaps, futurealternative posed by c

Conict of

Authorsinterest rel

Reviewers

Xu ZheMetabolic Houston, T

Dr ZakiInstitute of

Acknowled

The authand Researversity, Riy

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, et al. Depletion of L-asparagine supply and apoptosis ofia cells induced by human glycosylasparaginase. Leukemia3(6):116771.

es

inav Shrivastava is working as Assistant Pro-ollege of Life Sciences, Cancer Hospital and

nstitute, Gwalior, India. His primary researchludes the development of L-asparaginase fromsources in order to develop less toxic enzyme for-e is involved in the isolation of L-asparaginase

, animals, and microorganisms.

ul Arif Khan is an Assistant Professor in Collegey, King Saud University, Riyadh, Saudi Arabia.

t research focus is on cancer associated bacterialand their role in cancer aetiology and diagnosis,nt of anticancer substances from microbial origin,

system biology.

hsin Khurshid is a Lecturer at Directorate ofciences, Government College University, Faisa-istan. Previously he has served as Lecturer inPharmacy, King Saud University, Riyadh, Saudis research interest includes the bacterial Patho-tibiotic resistance mechanisms among bacteriatential role of bacteria in cancer diagnosis andnt.

d Abul Kalam has completed his PhD (Pharma- June 2011 from Department of Pharmaceutics,

Pharmacy, Jamia Hamdard, New Delhi, India.ember 2011, he is working as Assistant Profes-artment of Pharmaceutics, College of Pharmacy,

University, Riyadh, Saudi Arabia. His researchludes the development of nanoformulations as a

delivery system.

hir K Jain is working as Associate Professorent of Microbiology. Vikram University, Ujjain,research activities are focused on identificationug targets for new drug development, secondaryproduction from Keratinophilic and soil fungi.

deep K Singhal is a Professor in Depart-iological Science, Rani Durgavati University,dia. His research interest includes environmentalgy and development of anticancer formulations-asparaginase.1.002

Recent developments in l-asparaginase discovery and its potential as anticancer agent1 Introduction2 Mechanism of l-asparaginase activity3 Current asparaginase formulations: a comparative evaluation3.1 Current asparaginase formulations3.1.1 Native E. coli asparaginase3.1.2 Erwinia asparaginase3.1.3 PEG-asparaginase

4 Problems associated with clinical application of bacterial asparaginase4.1 Hypersensitivity4.2 Coagulation disorder4.3 Pancreatitis, hyperglycaemia, hepatotoxicity4.4 Resistance to l-asparaginase

5 Advancement in l-asparaginase discovery from alternative sources5.1 Algal asparaginase5.2 Plants asparaginase5.3 Fungal asparaginase5.4 Actinomycetes asparaginase5.5 Entrapment of l-asparaginase in erythrocytes

6 Conclusion and future directionConflict of interestReviewersAcknowledgementReferences

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ONCH_1928 - Recent Developments in L-Asparaginase Discovery and Its Potential as Anticancer Agent

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