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Treatment of Acute Lymphoblastic Leukemia : L-Asparaginases + PEG-L-
Asparaginases (Oncaspar®®/pegaspargase)
By: Brian Hernandez
What is ALL?
Acute lymphoblastic leukemia (ALL) is a form of leukemia in which immature white blood cells (“blasts”) become malignant and proliferate excessively in the bone marrow.
High number of blasts crowd healthy mature cells and prevents their proliferation.
Blasts can enter into the periphery and become metastatic and may form solid tumors in other tissues. CNS
Testicles Organs
Chest
Leukopenia Frequent infections Fevers Chills
Anemia Fatigue Weakness
Thrombocytopenia Bleeding Bruising Swollen/Bleeding gums Petechia
Other Loss of Apetite Bone/Joint pain Weight loss
Signs and Symptoms
Diagnosis of ALL
The most common diagnostic tool is a blood smear and total count
If ALL is suspected, a bone marrow biopsy and lumbar puncture may be performed to see the extent of the cancer.
ALL Facts and Figures
The American Cancer Society predicts that there will be 5,730 new cases of ALL reported in 2011.
75% of leukemia cases in children and adolescents (0-19) are ALL – the most common cancer in children under 20.
Treatment of the disease has improved but L-asparaginase (L-ASNase) continues to have a high rate of immunogenicity.
L-ASNases
Interest in this enzyme as a therapeutic began in the 1960’s when guinea pig serum showed to have anticancer properties.
It was later discovered that guinea pig serum has heightened levels of circulating L-ASNase.
L-ASNases have been found in many bacteria, yeast, molds, plants and in the plasma of certain vertebrates.
Structure of L-ASNases
Active forms L-ASNases in bacteria
exist as homotetramers Each monomer has ~333 AAs
Three commonly used L-ASNases Native form produced from E. coli (Elspar®) Native form produced from E. chrysanthemi Polyethylene glycol (PEG) modified E. coli L-ASNase
(Oncaspar®)
PEGylation
Refers to the covalent addition of at least one PEG chain to a molecule.
PEGylation improves protein drugs but may be associated with loss of bioactivity.
Mechanism of Action Against ALL Cells
Mechanism Continued
L-ASNases catalyze the hydrolysis of L-asparagine (Asn) to aspartic acid and ammonia.
Healthy human cells have sufficient concentrations of asparagine synthase. They do not rely solely on extracellular Asn for proper function.
Leukemic blasts do not have sufficient concentrations of the enzyme and cannot up regulate its expression when extracellular [Asn] gets low.
Lack of Asn inhibits protein synthesis in blasts and causes them to undergo apoptosis (cell cycle arrest at G1 phase).
Oncaspar®/pegaspargase
Oncaspar® is a PEGylated version of the native E. coli L-ASNase.
L-ASNase is a foreign protein and commonly induces the production of antibodies (58%) and sometimes severe allergic reactions (24%, 29%)
Oncaspar® has 5000-dalton units of monomethoxypolyethylene glycol conjugated to the enzyme.
The PEG groups on Oncaspar®
Reduce its immunogenicity Lower incidence of silent antibodies Increase t1/2 of the drug – reduce antibody mediate rapid clearance
Maintains the safety profile of native L-ASNase More convenient – lower doses and less frequent administration
Oncaspar®/pegaspargase
Oncaspar® is manufactured by Sigma-Tau who acquired it from Enzon.
The native E. coli L-ASNase is produced and isolated from E. coli and provided to Sigma-Tau by Lundbeck (makers of Elpar®).
The enzyme is then PEGylated using the Enzon’s Customized Linker Technology platform Enzon is payed a 5-10% royalty for the sale of Oncaspar®.
FDA Approval & Clinical Trial
Oncaspar® first approved by the FDA in February of 1994 for the treatment of patients with ALL who suffered sever immune responses to native E. coli L-ASNase.
The drug later received FDA approval in July of 2006 for first-line treatment of ALL in multiagent chemotherapy regimens.
Phase 1 Clinical Trial + Results
Patients 31 individuals treated
Treatment Patients received doses of pegaspargase
ranging from 500 to 8000 IU/m2
Drug was delivered IV over 1 hour for every 14 days Results – based on 27 evaluable patients
Disappearance of PEG-L-ASNase from plasma with an average t1/2 of 357 hours* (14.8 days) – biweekly administration. *t1/2 significantly different than future studies.
Concentration of PEG-L-ASNase in plasma, after the first IV infusion and at day 14, proved to be proportional to the administered dose.
Rate of total clearance of PEG-L-ASNase found to be 128 mL/m2/day compared to E. coli L-ASNase 2196 mL/m2/day.
Proved dosage of 2000-2500 IU/m2 is sufficient every 14 days for depletion of ASN in most patients
3 patients (11%) suffered anaphylaxis:
1 at 500 IU/m2
1 at 2000 IU/m2
1 at 4000 IU/m2
Phase 1 Clinical Trial: Results
Hypersensitivity toxicity showed no correlation to dosage
Phase 2 Clinical Trial
Patients 148 patients (93 M, 55 F)
≤ 20 years old B-precursor ALL
Treatment Reinduction with:
PEG-ASNase either weekly or biweekly (at random) at 2500 IU/m2 (IM)
Combination drugs following standard treatment administered
(doxorubicin, prednisone, and vincristine)
Evaluations CBC – 2 times per week. Serum alanine aminotransferase (ALT), total and
direct bilirubin, albumin, glucose, amylase, lipase, and plasma fibrinogen – days 1, 15, and 29.
Bone marrow aspiration – days 15 and 29 Serum L-ASNase concentration – weekly on days 8,
15, 22, and 29 Serum samples for E coli L-ASNase and PEG-L-
ASNase antibodies – weekly on days 8, 15, 22, and 29
Phase 2 Clinical Trial
Analysis L-ASNase enzyme activity measured by coupling L-ASNase
activity to oxidation of NADH to NAD+ (by alpha-ketoglutarate/oxaloacetate) and reading reactions at 340nm.
Serum anti-L-ASNase antibodies assayed using an antibody capture ELISA.
Phase 2 Clinical Trial
Stronger association between dosing schedule in patients with BM involvement
Weekly drug administration tended to show higher incidence of complete remission and lower incidence or disease resistance.
Phase 2 Clinical Trial: Results
*All patients with isolated extramedullary relapse achieved complete remission.
6 patients had allergic reactions to the drug (1 – hypersensitive) 3 out of the 6 were assigned
to the weekly PEG-ASNase arm.
3 patients suffered CNS events possibly associated with PEG-L-ASNase The 3 patients were assigned
to the weekly PEG-ASNase arm.
Phase 2 Clinical Trial: Results
Infections were common and resulted in the death of 4 patients. Unrelated to drug
High titer of antibodies against E. coli ASNase or PEG-ASNase correlated with low ASNase levels.
Phase 2 Clinical Trial: Results
Weekly PEG-ASNase regimen may have similar toxicity to biweekly administration.
But it may be more effective.
Adverse events were as common as the 2% to 3% incidence documented in other studies using E. coli ASNase in treatment.
More frequent administration of the drug maintained higher PEG-ASNase concentrations in plasma which is associated with lower antibodies.
Lower antibodies correlate with increased complete remission
Phase 2 Clinical Trial: Results
Phase 3 Clinical Trials
Patients 118 children (1-9) with standard risk
ALL
WBC ≤ 50,000/μL
Patients with massive lymphadenopathy, massive splenomegaly, large mediastinal mass, concurrent CNS, or testicular leukemia eligible
Treatment 4 weeks of induction
4 weeks of consolidation
2 8-week interim maintenance phases
2 8-week delayed intensification (DI) phases
Maintenance phase Duration was 2 (girls) and 3 (boys)
years
Patients also received the standard combination chemotherapy drugs (Vincristine, Prednisone, Cytarabine, Methotrexate, etc…)
Patients were randomly assigned to receive either 2500 IU/m2 of Oncaspar®
intramuscularly (IM) on day 3 of induction and each DI phase or
6000 IU/m2 of native E. coli ASNase IM 3 times per week, for 9 doses in induction, and 6 doses in each DI phase.
Patient Monitoring Physical exam
Blood and urine tests
CBC, creatinine, bilirubin, AST, ALT, urine glucose
Spinal taps
Bone marrow aspirates
Analysis L-ASNase enzyme activity
measured by quantifying ammonia produced from ASN – read by ELISA.
Serum anti-L-ASNase antibodies assayed using a modified indirect solid-phase ELISA.
AAs asparagine, aspartic acid glutamic acid and glutamine quantified via high-performance liquid chromatography.
Phase 3 Clinical Trials
Oncaspar® showed better clearance of blasts in ALL infected bone marrow than the E. coli L-ASNase.
Phase 3 Clinical Trials: Results
Oncaspar® showed better clearance of blasts in ALL infected bone marrow than the E. coli L-ASNase.
Oncaspar Day 7 Oncaspar Day 14 E. coli L-ASNase Day 7
E. coli L-ASNase Day 14
0
20
40
60
80
100
120
63
96
47
83
23
4
241014
0
29
8
Bone Marrow Status During Induction
M1 (<5% blasts) M2 (5%-25% blasts) M3 (>25% blasts)
% P
atie
nts
Phase 3 Clinical Trials: Results
The study sought to show a decrease in anti-L-ASNase antibodies when Oncaspar® was used.
This graph shows that pegaspargase significantly reduced the amount of antibodies produced. *Ratio reflects the amount of antibodies in
patients’ sera over the amount of antibodies in healthy individuals’ sera (- control)
Phase 3 Clinical Trials: Results
Oncaspar® has a longer t1/2 than E. coli L-ASNase (5.5 vs. 1.1 days).
Greater L-ASNase concentration though dosage and frequency of administration was lower.
Oncaspar® shows clinically significant enzymatic activity longer
Oncaspar® showed similar correlation between clearance of AAs in serum and CSF when compared to E. coli L-ASNase.
Note: Oncaspar® was being delivered less frequently and at lower doses.
Gln
Asn Asn
Gln
*Data collected during induction
Phase 3 Clinical Trials: Results
Production of anti-L-ASNase antibodies cause severe/life threatening allergic reactions and can silently decrease the efficacy of the drug, preventing its further use.
Oncaspar®, when compared to E. coli L-ASNase, showed (“why it’s better”): Lower production of allergic response initiating
and enzyme inactivating antibodies Better clearance of blasts from infected patients’
bone marrow More persistent and higher L-ASNase activity Similar, if not better, toxicity and efficacy
Longer t1/2 (by 4+ days)
The need for less frequent administration and lower doses of the drug – 1 administration can replace 6 to 9 administrations of E. coli L-ASNase.
Better EFS probability (88% vs. 85%)
Pharmacoeconomic component of the trial showed patient costs of Oncaspar was similar* to E. coli L-ASNase. *Later studies claim that the overall
costs associated with treatment using pegaspargase would be “considerably less” compared to therapies using conventional L-ASNases.
Phase 3 Clinical Trials: Results
Industrial Perspective
Sigma Tau has another ALL drug in Phase III clinical trials called EZN-2285 (calasparagase pegol)
Future Treatments: Mesenchymal cell disruption in bone marrow + ASNase
treatment Modifying ASNase to alter glutimase activity – reduce
side effects but have greater effect against cancer cells Modifications:
Entrapment of enzyme in liposomes/microcapsules/RBC Covalent coupling to macromolecules (dextran, albumin, …) Immobilization on polyacrylamide or agarose
Sales and Cost
Reported sales for Oncaspar®: $38.7 million in 2007. $50.1 million in 2008 $52.4 million in 2009
Sigma Tau upgraded production process in 2011 Spent $50 million dollars to avoid drug shortage Cost of Oncaspar® jumped from $2,625 to $5,670 per
vial Could be as high as $40k for full treatment. With government programs like Medicaid – $70/$490
Sales and Cost
Main competitor is Elspar® (Lundbeck) Cost is estimated at $190 per vial Administration is more frequent Oncaspar® shown to be equivalent to 9 doses Considering costs of complications and office visits it has
been estimated to not save much, if any, money. Sigma Tau Outreach Service (S.O.S.)
Assists patients by reimbursing patients’ copays 100% No limitations on family income or financial status S.O.S program provides Oncaspar® free of charge to
eligible patients who may not have insurance coverage or the ability to pay.
Patents
Patent Number – Date Patent Title Inventors/Assignee
4002531 – Jan. 11, 1977 Modifying enzymes with polyethylene glycol and product produced thereby
Pierce Chemical Company
4179337 – Dec. 18, 1979 Non-immunogenic polypeptides Davis; Frank F.
4729957 – Mar. 8, 1988 Process for manufacture of L-asparaginase from erwinia chrysanthemi
The United States of America as represented by the Department of Health
6087151 – Jul. 11, 2000 DNA coding for mammalian L-asparaginase
Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo
6113906 – Sep. 5, 2000 Water-soluble non-antigenic polymer linkable to biologically active material
Enzon, Inc.
6566506 – May 20, 2003 Non-antigenic branched polymer conjugates
Enzon, Inc.
7365127 – Apr. 29, 2008 Process for the preparation of polymer conjugates
Enzon Inc.
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