ind. sem. - all oncaspar

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


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.


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.


*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.


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.


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 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


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)


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


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.


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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