CENTER FOR DRUG EVALUATION AND RESEARCH
`
`APPROVAL PACKAGE FOR:
`
`APPLICATION NUMBER
`
`21-462
`
`Pharmacology Review(s)
`
`

`

`PHARMACOLOGY/TOXICOLOGY REVIEW AND EVALUATION
`
`NDA:
`Review number:
`
`,
`
`Sequence number/date/type of submission:
`Information to sponsor:
`Sponsor:
`
`1
`
`Manufacturer for drug product:
`
`21—462
`1
`
`000/ 1 0/25/02/NDA
`Yes(x), No( )
`Eli Lilly and Company.
`Indianapolis, IN 46285
`
`Eli Lilly and Company.
`Indianapolis, IN 46285
`
`Reviewer name:
`Division name:
`HFD #:
`
`Doo Y. Lee Ham, Ph. D.
`Division Oncology Drug Products
`HFD-ISO
`
`‘
`
`Review completion date:
`
`August 14, 2003
`
`Drug:
`
`Trade name:
`Generic name:
`Code name:
`CAS number:
`
`Chemical name:
`
`Molecular formula:
`Molecular weight:
`Master file #:
`Structure:
`
`ALIMTA (Pemetrexed for Injection)
`Pemetrexed disodium (MTA, LY231514)
`LY231514
`137281-23—3
`
`N-[4-[2-(2-Amino—4,7-dihydro~4-oxo-lH-pyrrolo [2,3-d]
`pyrimidin-S-yl) ethyl] benzoyl]—L-glutamic acid
`‘ disodium salt
`
`C20H19N506-2Na
`579. 49
`Not provided
`
`8
`90,14;
`
`n
`
`CO’N‘
`
`0
`
`
`
`
`
`NH
`
`m .
`3,144
`N
`
`Relevant IND:
`
`IND 40,061 (LY231514)
`
`Drug Class:
`
`A Thymidylate synthase inhibitor
`
`Indieation:
`
`Malignant pleural mesothelioma
`
`Clinical Formulation: Alimta (Pemetrexed Disodium for Injection) 500 mg/vial is supplied as a
`$—\
`'
`powder for reconstitution for intravenous infusion.
`
`Route of Administration:
`
`Intravenous Infusion
`
`

`

`Proposed use:
`
`Alimta (Premetrexed Disodium) is a folate antagonist proposed for the
`treatment of malignant pleural mesothelioma in combination with
`cisplatin. The recommended dose ofALIMTA is 500 mg/mZ
`administered as an intravenous rapid infusion over 10 minutes once
`every 21 days followed approximately 30 minutes later by a 2 hr
`infusion of 75 mg/m2 cisplatin.
`
`Disclaimer: Tabular and graphical information is from sponsor’s submission unless stated
`otherwise.
`
`APPEARS THIS WAY
`
`0H GRIGWAL
`
`

`

`Executive Summary
`
`1.
`
`Recommendations
`
`A.
`
`Recommendation on Approvability:
`
`The non-clinical studies adequately support the use ofLY231514 (A-LIMTA), by the
`intravenous infusion for the treatment of malignant pleural mesothelioma.
`
`The pharmacology information in the application do support the applicant’s claims for
`the mechanism of action.
`
`B.
`
`Recommendation for Nonclinical Studies:
`
`No further toxicology information is necessary to support the indication of malignant
`pleural mesothelioma.
`
`II.
`
`Summary of Non-clinical Findings
`
`A.
`
`Brief Overview of Non-clinical Findings
`
`LY23lSl4 (ALIMTA®; MTA; pemetrexed disodium) is a novel pyrrolopyrimidine
`antifolate antimetabolite and a specific inhibitor of thymidylate synthase (TS).
`lt exerts its
`antifolate antineoplastic activity by disrupting crucial folate-dependent metabolic processes that
`are essential for cell replication. Many studies have shown that LY23lSl4 requires intracellular
`polyglutamation for its cytotoxic effect and these polyglutamates potently inhibit several key
`folate-requiring enzymes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR),
`and glycinamide ribonucleotide forrnyltransferase (GARFT).
`LY231514 has shown its antitumor activity as a single agent against NSCLC, head and
`neck, colon, and breast cancer. LY231514 has been evaluated in combination with other
`chemotherapeutic agents. In particular, studies with mesothelioma ell lines (NCI-2052 and
`MSTO-2l lH) showed greater effects when LY231514 was combined concurrently with cisplatin.
`In a single dose studies, LY231514 demonstrated low toxicity in both mice (MLD=>4722
`mg/mZ) and rats (MLD=>87l8 mg/mZ) but higher toxicity in dogs (MLD=>2000 mg/m2). Death
`was preceded by hunched posture, ataxia, piloerection, decreased food intake, and clonic
`convulsion. Six weeks repeat dose studies were conducted using daily, twice weekly or weekly
`i.p. doses in mice and i.v. doses in dogs. Higher daily doses were not tolerated in dogs for more
`than 3 weeks. Generally the daily dose schedule was more toxic than the weekly administration
`of much larger dose. Mice tolerated weekly i.p dose of 944 mg/m2 (close to twice the clinical
`dose) without death or clinical signs of toxicity; weekly i.v. dose of 2099 mg/mZ (about four
`times the clinical dose) killed two of six dogs. Across species, chronic dosing at higher doses
`causes decreased food consumption, mucositis, decreased red cell parameters, leukopenia,
`neutropenia, increased hepatic enzymes, and decreased electrolytes. Microscopic changes occur
`in the thymus, lymph nodes, GI tract and intestine (enteropathy, mucositis-dog), testis, bone
`marrow, and skin. Clinically, rash, nausea, diarrhea, asthenia, and leukopenia and neutropenia
`are dose-limiting.
`’
`Non-clinical studies were done to evaluate the effects of rescue agents (leucovorin and
`thymidine) for the treatment of severe toxicity because of LY231514. Coadministration of
`leucovorin, a reduced form of folate, reversed the toxicity and hematological alterations induced
`
`.
`
`

`

`by LY231514 treatment in dogs. Dogs given LY231514 combination with thymidine had no
`clinicopathologic alterations associated with administration of LY231514.
`LY231514 iv. doses of 0.3 mg/m2 caused testicular atrophy and reduced fertility.
`LY123514 is embryotoxic and teratogenic in mice at 0.6 mg/m2. Carcinogenicity studies have
`not been conducted. LY231514 caused no genetic damage in the standard battery of tests;
`however, LY231514 was clastogenic in the vivo micronucleus assay. Non-clinical irritation
`studies also show that LY231514 has potential to cause ocular and dermal irritation.
`The pharmacokinetics of LY23 l 514 was found to be similar in mice following both i.v.
`or i.p. administration. After single i.v. administration to mice, dogs and man, plasma
`concentration of LY231514 declined rapidly. The AUC values increased in a dose-dependent
`manner in all species; The elimination half-life was shorter in dogs and man compared to mice.
`Compound-related radiocarbon was rapidly distributed following an iv dose, however, tissue
`levels did not persist beyond 3 hrs. In both mice and dogs, the major route of elimination was via
`the kidney and the majority of the parent compound was excreted unchanged.
`
`.
`
`B.
`
`Nonclinnical Safety Issues Relevant to Clinical Use:
`
`The sponsor does not need to do further non-clinical studies to support the proposed
`indication.
`
`11].
`
`Administrative
`
`A. Reviewer signature:
`
`ISI
`
`Doo Y. Lee Ham, Ph. D.
`
`Pharmacology-Toxicology Reviewer
`
`Concurrence-
`
`I
`
`David Morse, Ph. D.
`Supervisory Pharrnacologist
`Non-Concurrence-
`
`B.
`
`Supervisor signature:
`
`C.
`
`ccrlist
`
`

`

`, OVERALL SUMMARY AND EVALUATION:
`
`Pemetrexed disodium (LY231514) is a structurally novel pyrrolopyrimidine antifolate
`antimetabolite, which is a specific inhibitor of thymidylate synthase (TS). Antimetabolites are
`cytotoxic drugs that are structurally similar to naturally occurring molecules by competing the
`sites that are necessary for the synthesis of purines, pyrimidines, and nucleic acids.
`The mechanism of action of pemetrexed is not fully understood. However, many studies
`have shown that LY231514 is taken into the cell via the reduced folate carrier (RFC) and
`membrane folate-binding protein (FBP) carriers. Once in the cell, LY231514 is rapidly
`polyglutamated by folypolyglutamate synthetase and trapped inside the cell where exerts
`its antiproliferative or cytotoxic activity. Both parent and polyglutamated LY231514
`behave as competitive inhibitors of several folate-dependent enzymes, including
`thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide
`ribonucleotide transferase (GARFT), which are the key enzymes for de novo nucleotide
`‘biosynthesis.
`
`Pharmacology:
`Pemetrexed has shown its antitumor activity against a variety of tumor types in
`preclinical models with human tumor cells in vitro. In vivo, pemetrexed exhibited
`efficacy in mouse xenografts models.
`In in vitro antiproliferative studies, pemetrexed was found to have antitumor activity
`against a broad range of tumor cell lines, including leukemia (CCRF-CEM, L1210), lung (A549),
`mesothelioma (NCl-H2052 and MSTO-Zl 1H), breast (MCF7), colon (GC3 and HCT8), and
`ovarian cancer (SKOVl) cell lines. In the colony-forming assays, clinically relevant
`concentrations of LY2315 14 gave dose-dependent responses against a wide panel of specimens,
`including tumor types that were chemo-resistant to methotrexate, 5-FU, and raltitrexed.
`In in vivo efficacy studies, pemetrexed was active against human xenografts of colon YRCS,
`. breast MX-l, pancreatic BxPC-3, and lung LX-l carcinomas in nude mice. Also, pemetrexed
`was active against human colon xenografts (GC3 and VRCS), these tumors were sensitive to
`Lometrexol but resistant to Methotrexate.
`
`.
`
`LY231514 has demonstrated its activity not only as a single agent but also in
`combination with a variety ofother chemotherapeutic agents, including platinum (cisplatin,
`»' carboplatin), 5-FU, doxorubicin, CPT-l I, oxaliplatin, paclitaxel, and gemcitabine. In particular,
`studies with mesothelioma cell lines (MSTO-21 1H, NCl-H2052) showed synergistic effects when
`LY231514 was added simultaneously with cisplatin. In both NCI-H23 and NCI-H460 lung
`' Carcinoma cells, combination of LY2315 14 with cisplatin produced additive interaction,
`regardless of the sequence of administration.
`
`Pharmacokinetics:
`
`.
`
`.
`
`Pharmacokinetic studies ofLY231514 have been performed in mice and dogs. In mice,
`'plasma pharmacokinetics was compared following intraperitoneal doses of 20 and 200 mg/kg to
`that of intravenous dose of 20 mg/kg LY2315. The pharmacokinetics was determined in dogs
`. following single (7.5, 100 mg/kg) and multiple intravenous doses (0.11-104.96 mg/kg). The
`pharmacokinetics of LY23 1 514 was found to be similar in mice following intravenous or
`Intraperitoneal route. The terminal half-life ofLY231514 in mice was 7.8 and 10 hrs for 20
`_ mg/kg and 200 mg/kg intraperitoneal dose levels and 7 hrs for 20 mg/kg intravenous dose.
`After single intravenous administration to mice and dogs, plasma concentration of LY231514
`declined in a biphasic manner, a rapid distribution phase followed by a longer elimination phase.
`
`

`

`Studies Reviewed with This Submission:
`
`Pharmacology:
`Mechanism ofAction:
`NCPR l3:
`
`'
`
`NCPR 16:
`
`NCPR 18:
`
`NCPR 20:
`
`NCPR 28:
`
`NCPR 29:
`
`NCPR 30:
`
`NCPR 31:
`
`’ NCPR 02:
`
`NCPR 03:
`
`NCPR 04:
`
`NCPR 05:
`
`NCPR 12:
`
`NCPR11:
`
`- NCPR 32:
`
`HNCPR4m‘
`
`.NCPRO&
`
`NCPR19:
`
`Transport of LY231514 Disodium by both Reduced Folate Carrier (RFC) and
`Membrane- Folate Binding Protein (mFBP). Vol. 1.4, pages 1-16. 1995
`Mechanism of Transport of LY231514 Disodium and cross-resistance pattern in cells
`with markedly Impaired Transport of MTX. _Vol. 1.4, pages 1-12. 2000
`Effect of LY23 l 514 Disodium on Purified Folate-Requiring Enzymes and on the
`Proliferation ofDifferent Tumor cell lines in culture. Vol. 1.4, pages 1-1 1. 1997
`Dipyridamole potentates in Vitro activity of LY231514 Disodium by inhibition of
`Thymidine transport. Volume 1.4, pages 1-9.
`Potential Role of Dihydrofolate Reductase (DHFR) Amplification on Cellular
`Resistance to LY231514 Disodium. Vol. 1.5, pages 1-1 1.2001
`Role of Folic Acid in Modulating the Toxicity and Efficacy of LY231514 Disodium.
`Volume 1.5, pages 1-7. 1998
`LY231514 Disodium and Vitamin Supplements in the Human MX-l Breast
`carcinomas. Volume 1.5, pages ~1-12. 2002
`The Impact ofp53 status on Cellular Sensitivity to LY231514 and other Antifolate
`Drugs. Volume. 1.5, pages 1-13. 2001
`In Vitro Efficacy Studies:
`NCPR 01:
`Activity of the Multitargeted antifolate LY231514 in the Human tumor
`Cloning assay. Volume 1.3, pages 1—8.
`Effect of LY231514 Disodium and Cisplatin on Human mesothelioma cell lines.
`Volume 1.3, pages 1-15.
`'
`Effects of Cisplatin/LY231514 Disodium combination in non-small cell lung cancer
`cell lines. Volume 1.3, pages 1-22.
`Effects of Carboplatin and VLY2315 14 combinations growth of three human ttimor
`cell lines. Volume 1.3, pages 1-62.
`Effects of Cisplatin, Paclitaxel, and LY2331Disodium combinations in human
`carcinoma cell lines. Volume 1.3, pages 1-59.
`Cell cycle effects of LY231514 Disodium on Gemcitabine antitumor activity in HT~
`29 colon carcinoma cells. Volume 1.4, pages 1-9.
`Phase I and pharrnacologic study of sequence of Gemcitabine and the Multitargeted
`Antifolate agent in patients with advanced solid tumors. Vol. 1.4, pages l-l3.2000
`Role ofFolic acid in modulation the toxicity and efficacy of LY231514 Disodium,
`Volume 1.5, pages 1-7.
`Interaction of Pemetrexed Disodium (Alimta, Multi Targeted Antifolate) and
`Irradiation in Vitro. Vol. 1.4, pages 1-35.
`'
`Sequence dependence of Alimta (LY231514, MTA) combined with Doxorubicin in
`ZR-75-1 Human breast carcinoma cells. Volume 1.4, pages 1-1 1.
`In Vivo Efficacy Studies:
`NCPRO&
`Effects of Oxaliplatin and 5-FU/LY231514 Disodium combinations on growth
`inhibition of wild type and 5-FU-resistant HT-29 cells. Volume 1.3, pages 1-8.
`In Vivo antitumor activity ofLY231514 Disodium against various human tumor
`xenografts. Volume 1.4, pages 1-10.
`Role of Thymidylate Synthase in the Antitumor Activity of LT231514 Disodium.
`Volume 1.4, pages 1-9. 1999.
`.
`Safety Pharmacology:
`
`NCPR 29:
`
`

`

`Gen Pharm 1:
`In Vitro Studies ofLY231514—Na2 in the smooth and cardiac muscles of SD rats and
`
`Hartley albino guinea pigs. Volume 1.5, pages 1-36.
`Gen Pharm 3:
`
`The Acute Behavioral Effects of LY231514-Na2 following IV administration in
`Male CD- 1 mice Volume 1 5, pages 1-44.
`Gen Pharm 4:
`
`The Acute Effects of LY231514-N212 on gastrointestinal motility following IV
`administration in male CD-1 mice. Volume 1.5, pages 1-28.
`Gen Pharm 5:
`
`A Renal Pharmacology Study in female F ischer-344 rats given a single IV injection
`ofLY23151-Na2. Volume 1.6, pages 1-54.
`
`.
`j
`- Pharmacokinetics:
`ADME Report 1: Relative Bioavailability of IP administration and Plasma Pharmacokinetics of
`LY231514 in Male CD-l Mice after IV administration of 20 mg/kg or IP
`administration of 20 or 200 mg/kg (BE) LY231514 N32. Volume 1.6, pages 1-54.
`ADM E Report 2: Plasma Pharmacokinetics of LY231514 in beagle dogs after IV
`administration of 7.5 or 100 mg/kg (BE) LY231514 Na; (Toxicology Study D05091).
`Volume 1.6, pages 1-54,
`ADME Report 3:
`Summary of the Whole-Body Autoradiographic distribution of [”C]
`LY231514 Na; in CD-1 mice. Volume 1.6, pages 1-18. 1993
`ADME Report 5: Excretion and Metabolism of [”C] LY231514 Na; in male CD-l Mice after
`a Single IV dose of 20 mg/kg comparison with a Single Oral 20 mg/kg dose. Volume
`17 pages 116. 1993
`ADME Report 6: Excretion and Metabolism of [”C] LY231514 Na;in Female Beagle dogs
`after a single IV dose of7. 5 mg/kg and 100 mg/kg. Volume 1 7, pages 1-18.1993
`‘_ ADME Report 7:
`Protein Binding of l4C-LY231514 in mouse, dog and Human plasma.
`Volume 1.7, pages 1-8.
`ADME Report 8: Urinary Metabolites of [”C] LY231514 Na; in mice and dogs. Volume. 1.7,
`pages 1-16.
`ADME Report 9: Quantitative Whole-Body Autoradiographic Disposition of MC-I.Y231514
`Disodium in Male CD- 1 mice after a Single IV Administration of 20 mg/kg dose
`(Free acid) Volume 1 7, pages 1-.27
`ADME Report 10: Identification ofa Urinary Metabolite of[”C] LY231514 Na;in mice and
`dogs. Volume 1-. 7, pages 1 10.
`ADME Report 11:
`In Vitro interaction of LY23 1 514 with Human Cytochromes P450 CYP3A,
`CYP2D6, CYPP2C9, and CYP1A2 Volume 1. 6, pages 1-17.
`'
`ADME Report 12: Pharmacokinetic Interaction Study of LY231514 and Aspirin in beagle dogs
`following a single IV bolus of 25 mg/kg. Volume 1.6, pages 1-24.
`ADME Report 13: Pharmacokinetic Interaction Study of LY231514 and Ibuprofen1n beagle
`dogs following a single IV bolus dose of 25 mg/kg Volume 1. 6, pages 1 -.24
`
`.
`
`-
`
`I Toxicology:
`TOX Report 10: A Subchronic Toxicity Study in Beagle Dog given LY231514 Na2 Daily,
`Twice Weekly and Weekly by Intravenous Injection for 6 Weeks. Volume 1.8, pages
`1-167.
`
`’ TOX Report 26: A Subchronic Toxicity Study in Beagle Dogs given 4 Weekly Intravenous
`Injection of LY231514 Na; followed by a 3-Week Recovery Phase. Volume 1.8,
`pages 1-103 and 1.9, pages 104-232.
`
`

`

`TOX Report 28: A 6-Month Repeat-Dose Toxicity Study in Beagle dogs given Weekly
`Intravenous Doses of LY231514 Na2. Volumes 1.9, pages 1-219, and 1.10, pages 220-280.
`
`Special Toxicology:
`TOX Report 18: A Special Study in Beagle Dogs administered LY231514 Na2 Intravenously
`on Days 0 'and 3 followed by Continuous Intravenous Infusion of Thymidine for 72
`_ hr. Volume 1.10, pages 1-60.
`
`Reproductive Toxicology:
`TOX Report 27: A Segment 1 Reproductive Toxicity Study of LY231514 Na2 administered by
`Intraperitoneal Injection to Male CD- 1 Mice. Volume 1 11, pages 1- 100.
`TOX Report 25: A Segment II Study of LY231514 Na2 administered Intravenously to Pregnant
`CD- 1 Mice. Volume 1.11, pages 1- 101.
`
`Studies Reviewed Previous Submission:
`
`,
`Pharmacology:
`Cytotoxic Effects of LY231514 on CCRF-CEM leukemia cells
`Efficacy Studies of LY231514 in thymidine kinase deficient murine lymphoma
`model
`
`Safety Pharmacologv:
`Gen Pharm 2:
`
`Cardiovascular and Respiratory Effects of 'LY2315 14—Na2 administered IV to
`anesthetized dogs. Volume 1.5, pages 1-65. 1992
`
`Pharmacokinetics:
`
`ADME Report 1: Plasma Phannacokinetics of LY2315 14 in Male CD-1 Mice after IV
`'
`administration of 20 mg/kg or IP administration of 20 or 200 mg/kg (BE) LY231514
`Naz, Volume 1.6, pages 1-54.
`ADME Report 2: Plasma Pharmacokinetics ofLY231514 in beagle dogs after IV
`administration of 7.5 or 100 mg/kg (BE) LY231514 Na; (from Toxicology Study
`D05091). Volume 1.6, pages 1—54.
`
`Toxicology:
`TOX Report 5: The Acute Toxicity of LY23 1514 Na2 administered Intravenously to CD-l
`Mice (M02692). Volume 1.7, pages 1-25.
`TOX Report 6: The Acute Toxicity of LY231514 Na2 administered Intravenously to Fisher
`344 Rats (RO3492). Volume 1.7, pages 1-19.
`TOX Report 7: The Pilot Toxicity Study of LY2315 l4 given Intraperitoneally to CD-l for 2
`weeks (M11090). Volume 1.7, pages 1-37.
`TOX Report 9: A Subchronic Toxicity Study in CD] Mice given LY231514 Na2 Daily, Twice
`'
`Weekly, and Weekly by Intraperitoneal Injection for 6 Weeks(M1539l). Volume 1.7,
`pages 1- 67.
`TOX Report 8:
`Intravenous Dose-ranging toxicity studies in beagle dogs given single or
`multiple doses of LY231514 for up to 2 Weeks(DO239l and D0349!) Volume 1.8,
`pages 1-80.
`TOX Report 10: A Subchronic Toxicity Study in beagle dogs given LY231514 Na; Daily,
`Twice weekly, and Weekly by Intravenous Injection for 6 Weeks (D05091).
`Volume 1.8, pages 1-167.
`Special Toxicology
`TOX Report 12: Leucovorin Rescue ofBeagle Dogs given Lethal Doses of LY231514 Na2.
`Volume 1.10, pages 1-46.
`Genotoxicity:
`
`

`

`TOX Report 1: The Effect of LY231514 Na2 on the Induction of Reverse Mutations in
`Salmonella Typhimurium and E. Coli Using the Ames Test. Volume 1.10, pages 1-
`38.
`
`TOX Report 2: The Effect of LY23 1514 N32 on the Induction of Forward Mutation at the
`HGPRT-‘r Locus of Chinese Hamster Ovary Cells. Volume 1.10, pages 1-42.
`TOX Report 3: The Effect of LY231514 Na2 on the In Vito Induction ofChromosomal
`Abberations in Chinese Hamster Ovary Cells. Volume 1.10, pages 1-36.
`TOX Report 4: The Effect of LY231514 N32 for 2 Consecutive Days on the Induction of
`’ Micronuclei in Bone Marrow ofICR Mice. Volume 1.10, pages 1-45.
`
`Studied n_ot Reviewed with This Submission:
`Pharmacology:
`
`(
`
`

`

`Reviewer: Doo Y Lee Ham, Ph.D.
`
`NDA 21—462
`
`1.
`
`PHARMACOLOGY
`
`Pemetrexed disodium (ALIMTA. MTA, LY231514) is a novel pyrrolopyrimidine
`antifolate antimetabolite and a specific inhibitor of thymidylate synthase (TS). It is a structural
`analog of Lometrexol, whichIS a potent inhibitor of glycinamide ribonucleotide formyltransferase
`(GARFT).
`The mechanism of action of pemetrexed is not fully understood. However, many studies
`indicate that TS is the locus of action of LY231514, and that TdR is able to reverse LY231514~
`induced cytotoxicity in vitro and in vivo. The enzyme thymidylate synthase (TS) is a folate-
`requiring'enzyme that catalyzes the transformation of dUMP to dTMP. This reaction requires, 5,
`10-methylenetetrahydrofolate, as a co-factor. Like MTX, LY231514 is folate-based TS inhibitor
`and competes with the co-factor for TS.
`Pemetrexed is a mild inhibitor of thymidylate synthase (TS) and dihydrofolate reductase
`(DHFR), while the polyglutamated forms of LY231514 are more potent inhibitors of TS, and
`mild inhibitors of DHFR and glycinamide ribonucleotide formyltransferase (GARFT).
`
`Enzyme Ihhieittbn by ALIMTA ln‘
`Comparison to Other Antimetabolites
`
`
`
`I”;
`
`..“mgf
`
`Imam.
`
`fréwj“,E
`w
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`n W
`AthA
`Wmuubma SYNTHESIS
`PuRmEégr‘Fmesls
`Mluu-
`My: ,—
`DNA
`DNA, RNA blocynfivonll
`AUMTA dlrocm Inhlbm both pyrimldlno and pull-o ”mu
`
`LY231514 is taken into the cell by both reduced folate carrier (RFC) and membrane
`folate-binding protein (FPB) transport systems. Once inside the cell, LY231514 is rapidly
`polyglutamated by folypolyglutamate synthetase (FPGS). Studies with partially purified proteins
`showed that LY231514 was an efficient substrate for FPGS. The rate constant (Vm/Km) for
`LY231514 in the FPGS catalyzed reaction was about 100 to 400 times greater than that for MTX
`(Habeck et al). The more rapid and efficient polyglutamation of LY231514 allowed it to
`accumulate in cells faster than MTX, that exerts its antiproliferative or cytotoxic activity. Both
`. parent and polyglutamated LY231514 behave as competitive inhibitors of several folate-
`dependent enzymes, which are part of the thymine and purine nucleotide biosynthesis pathways.
`AMP
`
`
`
`
`
`run-ovum
`”waif
`>~nov_uu~am_
`
`.5;
`-.
`
`10ch
`
`t
`5,1o—CH,~FH. fl .
`
`.
`
`DNA Synmuh
`
`

`

`Reviewer: Doo Y Lee Ham, PhD.
`
`NDA 21-462
`
`I Mechanism ofAction Studies .
`Table 1
`Parameters
`TestS stem 5
`
`Results
`
`Transport ofLY231514 by
`reduced folate carrier (RFC)
`and membrane folate.binding
`protein (FBP)
`
`
`
`Stud No.
`
`NCPR 13
`
`NCPR .16
`
`NCPR 18
`
`Cell lines expressing different levels of
`RFC & FBP:
`
`Human leukemia lines:
`CEM (RFC+), CEM/MTX (RFC—). &
`CEM-7A cells (RFC+++)
`
`LY2315l4 & 5-methyltetrahydrofolate had similar affinity for
`RFC (lC50=4 uM). LY231514 was ~l .5 fold more effective
`than folic acid in binding to FBP.
`
`ln CEM (RFC+) cells cultured in 2 uM folic acid, CEM/MTX
`(RFC), cultured in 2 uM folic acid, & CEM-7A cells
`(RFC+++) cultured in 0.2nM leucovorin, leo ofLY231514
`were 23, 470, & 5.2 nM. respectively.
`'
`
`Murine leukemia cell lines:
`L1210 cells (RFC+)
`Ll2l0-B73 (RFC+/FBP+++), &
`L1210-FBP(RFC-/FBP+++)
`
`ln L1210 cells (RFC+) grown in 2 uM folic acid, ICSO of
`LY231514 was 14 nM.
`ln L1210-B73(RFC+/FBP+++) grown in 1 nM leucovorin. 20
`nM leucovon'n or 1 nM leucovorin + 20 nM folic acid, lC50
`values ofLY23 l 514 were 4, 15, and 16 nM. respectively.
`ln LlZlO-FBP (RFC-IFBP+++) grown in 1 nM leucovorin, 20
`nM leucovorin or 1 nM leucovorin + 20 nM folic acid. 1C50
`values were 1.6, 5.6, and 162 nM. res ectivel
`.
`
`1.
`lnllux rate constant (Vmax/Kt) of LY231514 was 1.3 fold
`
`higher than MTX. Higher levels of LY231514 were due to
`more rapid polyglutamation of LY23 1 514 & lower rate of
`LY231514 efflux via RFC-independent mechanism compared
`to MTX.
`2.
`Two L1210 variants with point mutation in RFC showed
`cross-resistance to LY231514 dependent on media folate.
`ln
`folic acid, ICSO increased 5.8 — and & 22-fold for LY231514 &
`12- & 71-fold for MTX.
`ln 5~FTHF media, the lC50 increased
`only 3.2- & 2.4-fold for LY23l514, but 10.7- & l 1.4—fold for
`MTX. Sensitivity to both drugs showed an inverse relationship
`
`with folate 001.
`
`Murine leukemia cell lines:
`Mechanism oftransport of
`
`
`L1210
`LY231514 & cross-resistant
`
`
`MTX'A & L1210-Gm -MTX-resistant
`pattem in cells with markedly
`
`L1210 variants with point mutation
`impaired transport of MTX
`
`
`
`(single amino acid substitution)
`
`
`
`
`
`
`Effect of LY23|514 on
`Cell lines:
`
`Growth inhibition assays:
`
`
`l. LY231514 activity against CEM, 0C3, & HCT8cells was
`Wild type leukemia,CCRF-CEM (CEM)
`purified folate enzymes and
`
`
`prevented by 5 uM thymidine completely when LY231514 was
`& its polyglutamation deficient CR15
`on proliferation of different
`
`
`tumor cells in culture
`sublinc. colon carcinoma GC3/C1 .
`at ICSO in all cell lines. At higher concentration, drug effect
`
`was completely blocked by the combination of5 uM thymidine
`(6C3), ileocecal carcinoma HCT8, wild
`
`and 100 uM hypoxanthine.
`type breast carcinoma ZR-75-1 & its 3
`
`sublines AAR-FR+, MTXR, and
`2. In the polyglutamation defective CR15 cells, 1C50 of
`
`MTXR-BBB—FR+
`LY231514 increased >7800-fold compared to that for wild type
`
`CEM cells.
`
`
`
`

`

`Reviewer: Doo Y Lee Ham, PhD.
`
`'
`
`NDA 21-462
`
`Table 1.
`
`(Continued) _Mechanism of Action Studies
`
`NCPR l8
`
`Kinetic studies ofLY23l5l4
`
`
`
`NCPR 20
`
` Table l: Inhibition of Purified Palate-Requiring Enzymes by Alimta
`TS, DHFR, and GARFT were assayed
`
`
`
`_‘
`. and AICARFT
`Ki ValuesLn‘M, n23)
`___
`and its synthetic y-poly-
`
`TS
`DHFR
`GARFT
`
`
`
`
`
`glutamates (glu, and glus)
`inhibition was assayed by monitoring W
`
`
`
`
`
`930°
`7-0
`the formation of[6S]-5,6,7,8-
`“3m”
`'09
`
`
`
`
`380
`7.1
`tetrahydrofolale
`Altmta-(glug)
`LG
`_A_l_i_m___ta-(g|_Us1_ _ __l._3..__._._7..2_ __ $25 __.,
`
`
`5,l0-MTHF
`IO-FTHF
`
`dchydrogenasc smthetase AlCARFT
`9.0
`364
`3.58
`Alimta
`3.7
`25
`out
`Alimta-(gIUJ)
`
`
`AME-(211.15).
`LO
`L6
`0.26
`
`
`LY23 l 5l4 & its polyglutamated forms behaved as a competitive
`
`
`inhibitor with respect to folate cofactor in folate enzymes studies.
`
`
`Ki values ofLY23l5 l4 against TS. DHFR, GARFT & AlCARFT
`
`
`
`were l09, 7, 9300, and 3580 nM, res ectivel
`.
`'
`l) LY23l5l4 inhibited tumor growth & lCSO were -—28, -46. -52,-
`COR L23 lung cancer cell line;
`Dipyridamole potentiates in
`
`
`
`
`
`640 nM for COR L23, T47D, MCF7. & A549, respectively.
`A549 non small cell lung ca. cell line,
`vitro activity of LY23lSl4 by
`transport
`
`MCF7 & T47D breast ca. cell line
`2) Thymidine (l uM) completely prevented growth inhibition by
`inhibition of thymidine
`
`L23l5l4 at lC50 in all cell lines. At l0x lC50, growth inhibition
`
`was only partially reversed by thymidine (st uM), while both
`thymidine and hypoxanthine are required for complete reversal.
`
`3) Dipyridamole (l uM) inhibited thymidine transport 89% or more
`in all tested cell lines. whereas hypoxanthine transport was
`
`inhibited only in A549 & MCf7 cells. Dipyridamole (l uM)
`
`revented th midine/h
`oxanthine rescue of LY23l5l4.
`Potential role of DHFR
`In MTX-resistant CEM/R line, cells were 8-fold less resistant to
`CEM-parental human leukemia T-
`
`
`
`
`LY2315l4 than the selective DHFR inhibitor, MTX. LY23I5 l4
`lymphoblast cell line and two MTX-
`amplification on cellular
`
`
`
`
`
`resistance to LY2315 l4
`resistant sublines. CEM/R (resistance
`cytotoxicity was overcome by thymidine addition in contrast to
`
`
`
`
`due to increase in DHFR) & CEM/T
`combination ofthymidine & hypoxanthine required for protection
`
`
`
`in parental cells. However, this line was 54-fold resistant to
`(resistance due to drug transport)
`
`
`LY23 l 514 compared to parental cells. suggesting LY23 l5 l4
`
`targets DHFR as well as TS.
`In the MTX-deficient CEM/T line,
`
`
`cells were 91-fold resistant to LY23l514, 315 -fold resistant to
`
`
`
`raltitrexed, and 694-fold resistant to MTX
`Role of folic acid in
`CCRF-CEM (leukemia)
`Growth inhibitory lC50 values obtained in 2 nM folinic acid for
`
`
`
`
`lGROVI. KB, 6C3, LX-l, & CEM were 44, 34, 12. 4 & 4 nM.
`GC3 (colon)
`modulating efficacy ofthe
`
`
`
`
`lGROVl (ovarian)
`respectively. Folic acid (<l0 uM) in the media had little effect on
`multitargeted antifolate.
`
`KB e idermoid
`-rowth inhibito
`activit ofLY2315l4 in cell lines tested.
`
`LY23 l 514 on different cell
`
`
`
`
`
`
`NCPR 28
`
`NCPR 29
`
`
`
`
`
`

`

`*Reviewer: Doo Y
`
`Lee Ham, Ph.D.
`
`NDA 2l-462
`
`NCPR 30
`
`Effects of vitamin
`
`supplementation on activity of
`LY231514 in human MX-l
`breast carcinoma
`
`
`
`TG D=tu mor growth delay
`
`LX-l (human lung) carcinoma cell
`lines
`
`Nude mice bearing MX-l breast
`xenograf‘t received 11’ 100 or 150
`mg/kg doses ofLY2315|4 daily on
`days 7-11 and 14-18 alone or co-
`administered with nontoxic doses of
`either folic acid (6 or 60 mg/kg.
`orally), vitamin B6 (100 mg/kg, orally)
`or vitamin BIZ (165 mg/kg, lP)
`
`However, folic acid (100 uM) in the media raised [.Y23lfil4 lCSO
`values against IGROVI. KB, 0C3, LX-l. & CEM were 25-, 17-, 9.
`6-, & 22-fold. respectively. However. presence of folinic acid (10
`uM) in the media raised LY23I514 lC50 values against these cells
`>970-, 78-, 47—. 82-, & l30-fold, res ectivel .
`Tumor growth delay was measured (days) for the treated and for the
`controls .in the following table.
`
`Tumor Growth Delay 1 Days)
`I7
`.
`I7
`22
`17
`22
`21
`2I
`23
`21
`24
`
`Test Comgoundng/kg
`LY23l5l4 l00 alone
`+ folate 6
`+ folate 60
`+ Vitamin B6100
`+VitaminB12165
`LY23ISI4 150 alone
`+ folate 6
`+ folate 60
`+ Vitamin 86 100
`+ Vitamin BIZ I65
`Folate alone
`6
`60
`Vitamin alone
`5.7
`86 100
`12
`BIZ 165
`LY231514 alone delayed tumor growth by 17 to 21 days. The
`combination of LY231514 with folate 60 mg or vitamin I312 165
`increased TGD by 22-24 days.
`Vitamins or folic acid did not increase toxicity (as determined by
`body weight) or alter the antitumor activity of LY23 1 SM disodium
`in the human MX-I breast carcinoma.
`
`7
`12
`
`

`

`
`Reviewer: Doo Y Lee Ham, Ph.D.
`
`-
`
`'
`
`'
`
`NDA 21-462
`
`Table 2.
`
`In Vitro efficacy studies: '
`
`Stud No.
`NCPR 01
`
`NCPR 02
`
`NCPR 03
`
`NCPR 04
`
`NCPR 05
`
`
`
`
`
`
`
`
`
`v
`. Concentrations
`LY231514 concentrations at
`0.1. 1. and 10 ug/mL (0.2, 2.1,
`& 2| uM)
`
`CellT es
`
`
`Of358 specimens plated in the short 1 hr exposure studies, 148 (41%)
`
`Cells from multiple tumor
`Activity of LY23 1514
`
`
`
`were evaluable. Overall, in clinically achievable LY231514
`specimens (including
`against patient‘s tumor cells
`
`
`
`concentrations. responses were dose-dependent: response rates were
`colon, lung, breast. &
`in colony-forming assay
`
`
`
`
`mesothelioma) collected
`3% ofspecimens (4/144) at 0.1 ug/mL, 11% (17/148) at 1.0 ug/mL.
`
`
`
`from different patients
`and 23% (33/141) at 10 ug/mL. At 10 ug/mL, responses included
`
`
`
`undergoing routine
`colorectal cancer at 32% (9/28). NSCLC at 25% (6/24). & and
`
`
`
`diagnostic or therapeutic
`mesothelioma 66% (2/3). Scatter plots analysis demonstrated
`
`
`procedures
`LY231514 activity (multitargeted antifolate) was not completely
`
`
`
`cross-resistance with those ofcisplatin, S-FU. irinotecan, and
`
`aclitaxel.
`
`Effects of LY23 15 14 &
`'Mesothelioma lines:
`LY231514: 15 nM-2 uM;
`
`
`
`ln growth inhibition study, cells were exposed for 72 hr. The growth
`
`
`inhibitory lC50 for LY231514 in MSTO-Zl 1H, NCl-H28, and NCl-
`cisplatin on human
`MSTO-Zl 1H, NCl-H28,
`Cisplatin: 30 nM-lO uM
`
`
`
`
`mesothelioma cell lines in
`NCl-H2052
`.
`112052 human mesothelioma cells were 31, 180, & 209 nM,
`
`
`
`
`respectively.
`.
`MTT assay and Flow
`
`
`
`MSOT-Zl 1H Was chosen for the combination studies of LY23 1514
`cytometry assay
`
`
`and cisplatin. The lC50 for cisplatin was 1.33 uM for MSTO-21 111.
`
`Using simultaneous 72-hr exposure, LY231514 and cisplatin produced
`
`synergistic growth inhibitory activity using both constant and non-
`constant ratio techniques in MTT assays.
`
`DNA flow cytometry studies indicated that LY2315 14 causes a
`
`
`buildu ofcells near Gl/S interface after 24 h incubation.
`NCl-H23 adenoearcinoma
`LY231SI4: 11.7 nM-
`Growth inhibition: LY2315U4 when used in combination with
`Activity of LY231514 in
`
`
`
`
`
`combination with other
`ll7 uM;
`cisplatin produced an additive activity regardless of sequence of
`& NCl-H460 large cell
`
`
`
`administration. The [C50 values ofLY231514 and cisplatin in NC!-
`antitumor agents against
`carcinoma of the lung
`Cisplatin: 16.7 nM-167 uM
`
`
`
`
`
`
`
`various tumor cell lines
`HZ3 cells were 235.8 and 441.2 n_/mL, respecti_vcl
`.
`LY231514: 0.3 nM-30 uM;
`p NCl-460 (NSCLC)
`Activity ofLY231514 and
`Growth inhibitory effects of LY23 1 514 and carboplatin and were
`
`
`
`
`SKOV-3 (Ovarian cancer
`Carboplatin: 0.3-3000 uM
`additive when cells were exposed to both agents regardless of
`
`carboplatin against three
`
`
`
`human tumor cell lines
`HT29 (Colorectal cancer)
`sequence (MTA followed by carboplatin or carboplatin followed by
`
`
`
`
`MTA or simultaneous ex IOSUI‘C in althrec cell lines.
`
`LY231514: 20-200 nM;
`
`
`
`A549, human lung
`In LY231514 and cisplatin combination. simultaneous exposure to
`Schedule-dependent
`
`
`carcinoma (ca.)
`cytotoxic effects of
`LY231514 & cisplatin produced antagonistic effects in A549 and
`Cisplatin: 0.2-2 uM;
`
`
`
`
`MCF7 cells and additive effects in PAl and WiDr cells. Similar
`MCF7, breast ca.
`LY231514 and cisplatin of
`paclitaxel: 5 pM-IO nM
`
`
`
`
`
`
`PA], ovarian ca.
`effects were observed in LY231514 and paclitaxel combination.
`paclitaxel against various
`
`
`
`cell lines
`WiDr, colon ca.
`Sequential exposures to MTA followed by cisplatin or paclitaxel
`
`
`
`
`produce synergistic effects whereas simultaneous exposure had
`
`antagonistic effects. The present findings show that the interaction of
`
`
`
`MTA and cis latin or aclitaxel is schedule-de-endent.
`
`