Cancer Chemotherapy
`and Biotherapy:
`Principles anCf Practice
`SECOND EDITION
`
`EDITED BY
`
`Bruce A. Chabner, M.D.
`
`Chief, Hematology /Oncology
`Clinical Director
`Massachusetts General Hospital Cancer Center
`Boston, Massachusetts
`
`Dan L. Longo, M.D.
`
`Director, Biological Response Modifiers Program
`Division of Cancer Treatment
`National Cancer Institute
`Frederick, Maryland
`
`Lippincott - Raven
`
`PUBL I S HER S
`
`Philadelphia • New York
`
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`
`Copyright© 1996 by Lippincott-Raven Publishers. All rights reserved. This book is protected by copy(cid:173)
`right. No part of it may be reproduced, stored in a retrieval system, or transn;itted, in any form or by
`any means-electronic, mechanical , photocopy, recording, or otherwise-without the pnor wntten
`permission of the publisher, except for brief quotations embodied in critical articles and reviews. Printed
`in the United States of America. For information write Lippincott-Raven Publishers, 227 East Wash(cid:173)
`ington Square, Philadelphia, PA 19106.
`
`Library of Congress Cataloging-in-Publications Data
`
`Cancer chemotherapy and biotherapy : principles and practice / edited
`by Bruce A. Chabner, Dan L. Longo- 2nd ed.
`p. em.
`Rev. ed. of: Cancer chemotherapy.
`Includes bibliographical references and index .
`ISBN 0-397-51418-2 (hard: alk. paper)
`1. Cancer-Chemotherapy. 2. Cancer-Immunotherapy.
`(Dan Louis), 1949-
`II. Cancer chemotherapy.
`[DNLM: l. Neoplasms-drug therapy. 2. Biological Products-therapeutic use.
`3 . Antineoplastic Agents-therapeutic use. 4. Chabner, Bruce. QZ 267 C2 1515 1996]
`RC27l.C5C32219 1996
`616.99'4061-dc20
`DNLM/DLC
`for Library of Congress
`
`I. Longo, Dan L
`
`95 -38920
`C IP
`
`The material contained in this volume was submitted as previously unpublished material, except in
`the instances in which credit has been given to the source from which some of the illustrative material
`was derived.
`Great care has been taken to maintain the accuracy of the information contained in the volume. How(cid:173)
`ever, neither Lippincott- Raven Publishers nor the editors can be held responsible for errors or for any
`consequences arising from the use of the information herein.
`The authors and publisher have exerted every effort to ensure that drug selection and dosage set forth
`in this text are in accord with current recommendations and practice at the time of publication. How(cid:173)
`ever, in view of ongoing research, changes in government regulations, and the co nstant flow of infor(cid:173)
`mation relating to drug therapy and drug reactions, the reader is urged to check the package insert for
`each drug for any change in indications and dosage and for added warnings and precautions. This is
`particularly important when the recommended agent is a new or infi-equcntly employed drug.
`Materials appearing in this book prepared by individuals as part of tl1eir official duties as U.S.
`Government employees are not covered by the above-mentioned copyright.
`
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`
`Ca~tct:?' Cht:?notbt:rnp_l' a~td Biotht:rnp)'J sam1d aiitiou}
`edited by Bruce A. Chobncr ond Don L. Longo.
`Lippincott- Raven Publishers, Philadelphia © 1996
`
`CHAPTER
`
`'--
`
`Antifolates
`Edward Chu and Carmen J. Allegra
`
`donates its one-carbon group to tl1e reductive methylation
`reactio n converting deoxyuridylate (dUMP) to thymidy(cid:173)
`late (dTMP) (see Fig. 6-1). In addition to yielding a one(cid:173)
`carbon group, 5, 10-methylenetetrahydrofolate is oxidized
`to dihydrofolate, vvhich must then be red uced to tetra(cid:173)
`hydrofolate by the enzyme DHFR in order for it to rejoin
`the pool of active reduced folate cofactors. In actively pro(cid:173)
`liferating tumor cells, inhibition of DHFR by methotrex(cid:173)
`ate (MTX) (see Fig. 6-2) or other 2 ,4 -diamino folates may
`lead to an accumulation of tolates in the inactive dihydro(cid:173)
`folate form, witl1 variable depletion of reduced folates. 8- 1•
`Folate depletion, however, does not fi.11ly account for the
`metabolic inhibition associated with antifolate treatment,
`since the critical reduced folate pools are relatively pre(cid:173)
`served even in the presence of cytotoxic concentrations of
`MTX. Add itional factors may contribute to MTX-associ(cid:173)
`ated cytotoxicity, including metabolism of the parent
`compound to polyglutamated derivatives and tl1e accumu(cid:173)
`lation of dihydrotolate and 10-formyld.ihydrofolate poly(cid:173)
`glutamates as a consequence of DHFR i~1hibition 8•9• 1 5- 1 7
`MTX polyglutamates, dihydrofolate polyglutamates, and
`10-formyldihydrofolate metabolites represent potent direct
`inhibitors of tl1e folate-dependent enzymes of thymidylate
`and purine biosynthesis. 18- 23 Thus inhibition of DNA
`biosyntl1esis by 2,4-diamino folates is a multifactorial
`process consisting of botl1 partial depletion of reduced
`folate substrates and direct inhibition of folate-dependent
`enzymes. The relative role of each of these mechanisms in
`determining antifolate-associated metabolic inhibition may
`depend on specific cellular factors tl1at vary among difterent
`cancer cell lines and tumors.
`
`CHEMICAL STRUCTURE
`
`Various heterocyclic compou nds with the 2,4-diamino
`structural configuration have antifolate activity and include
`such as pyrimethamine and
`pyrimidine analogues
`trimethoprim20- 29 (see Fig. 6-2 ), classic pteridines such
`as aminopterin and methotrexate/ and compounds
`with replacement of the nitrogen at either the 5
`8, or 10 position witl1 a carbon atom, such as tl1e quina~
`
`109
`
`The folate -dependent enzymes represent attractive tar(cid:173)
`gets .for antitumor chemotherapy because of their critical
`role 1n the synthesis of the nucl eotide precursors of DNA
`(Fig. 6-1 ). In 1948, Farber et al. 1 were the first to show
`that aminopterin, a 4-amino acid analogue of folic acid,
`could 1nhibit the proliferation of leukemic cells and pro(cid:173)
`duce remissions in acute leukemia. Their findings ushered
`1n the era of antimetabolite chemotherapy and generated
`great interest in the antifolate class of agents. Si nce then,
`the r .
`b
`c H11cal value of antifolate compounds has
`een
`roven in the treatment of the leukemias, breast cancer,
`I
`·
`·
`lead
`d
`d
`'
`an
`neck cancer, lympho mas, an
`c 10nocaro-
`110111a2 Their clinical application also has been extended
`~~ the treatment of nonneoplastic disorders, including
`~ leu~natoid arthritis,3 graft-versus-host .disease following
`one marrow transplantation, 4 psonasis, 5 bactenal and
`P.lasmodial infections 6 and opportunistic infections asso-
`Ciated
`. h
`' .
`.
`d fi .
`d .
`(
`W1t
`the acqmred Immuno e c1ency syn 1 ome
`bAIDS) _7 It is fair to state that this class of agents is the
`lest understood and most versatile of all the cancer
`C1enlotherapeutic drugs (Table 6-1 ).
`
`P 1
`
`
`
`MECHANISM OF ACTION
`
`1
`·
`·
`Substit
`_
`. ~It1on of an amino group for the hydroxy at tl1e
`4
`position of the pteridine ring is the critical change in
`h
`·
`l
`t e str
`d
`. ucture of antifolate compounds tl1at lea s to t 1e1r
`~ntltumor activity (Fig. 6-2). This change transforms the
`~1~lecu]~ fi·om a substrate to a tight-binding inhibitor of
`~111Ydrotolate reductase (DHFR), a key enzyme in intra-
`I .
`. .
`ce ular c I
`f.
`•o ate homeostasis. The cnt1ca
`Importance o
`b}{ '
`a . FR stems from the fact that folic acid compounds are
`I d
`Ct:Jve as ·
`·
`·
`' coenzymes only m their fully reduced teu·a 1y ro-
`f,
`1
`P~aat~ f~rm. There are two specific te~·ahydrofolates that
`. Y ssenual roles as one-carbon earners mvolved 111 the
`synthes·
`f
`·
`. 15 o DNA precursors. 10-Formylteu·ahydrofolate
`.
`10Vldes it ·
`P
`·
`tl
`f
`.
`s one-carbon group for tl1e de novo syn 1es1s o
`P
`unnes in
`·d
`·b
`·
`·
`·
`·
`.
`nu
`1eacuons mediated by glycmeam1 e n o-
`1
`bo ~eo tide (GAR) ti·ansformylase and aminoimidazole car(cid:173)
`on~amide ribonucleotide (AI CAR) transformylase. A sec-
`cofactor, 5, 10-metl1ylenetetrahydrofolate (CH2FHo~),
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`
`110
`
`CANCER CHEMOTHERAPY AND BIOTHERAPY
`
`Figure 6· 1. Sites of action of MTX, its polyglutamated metabolites [MTX(Giun)], and folate by-products of the inhibition of DHFR, in·
`eluding dihydrofolate (FH2) and 1 0-formyldihydrofolate ( 1 O-CHO·FH2). Also shown are 5,1 O·methylenetetrahydrofolate (CH2·FH.),
`the folate cofactor required for thymidylate synthesis, and 1 0-formyltetrahydrofolate ( 1 O·CHO·FH4), the required intermediate in the
`synthesis of purine precursors. (From Chabner eta\. In: DeVita VT, Hellman S, Rosenberg SA, eds. Cancer principles and practice of
`oncology. Philadelphia: JB Lippincott, 1989:349.)
`
`5-CHO -FH 4
`(Leucovorin)
`
`dUMP
`
`\
`
`dTMP
`
`,Reductase
`
`' '
`.......... ' ..... ~
`
`......-;:;_--!_.~ 10-CHO-FH2
`
`Compound
`
`Inhibits
`
`MTX
`
`Dihydrofolate reductase
`
`MTX(Giun)
`
`Dihydrofolate reductase
`Thymidylate synthase
`AICAR transformylase
`GAR transformylase
`
`Thymidylate synthase
`AICAR transformylase
`GAR transformylase
`
`{
`
`10-CHO-FH2 (Giun)
`
`{
`
`Thymidylate synthase
`GAR transformy\ase
`
`zolines ( trimetrexate, piritrexim )2+-26 and l 0-ethyl- l 0-
`( l 0-EDANl , Edatrexate ). 27 Com(cid:173)
`deazaaminopterin
`pounds with preservation of the benzoylglutamate termi (cid:173)
`nal group are transported by a folate-specific system in the
`cell membrane, whi le those lacking a terminal glutamate
`do not require active transmembrane transport and have
`activity against MTX -resistant cells that lack the folate
`transporter. 28 Recently, investigators have designed anti(cid:173)
`fo late analogues directed at targets other than DHFR,
`including t hose folate-dependent enzymes required for
`
`the de novo synthesis of purin es and thymidylate. A host
`of potent thymidylate synthase inhibitors such as 1 0-pro·
`pargyl-5,8-dideazafolate (PDDF, CB3717j29 and close!{;
`related compounds ZD1694,:~o LY2315l4,31 1843U89,
`and 5,8-dideazatetrahydrofolic acid (DDATHF )/3 an !11"
`d re(cid:173)
`hibitor of GAR transformylase, have been develope
`cently. Although each of these analogues has unique srruc -_
`tural features distinct from MTX with eq ual or greatel
`. I .b. .
`d
`, 1dent
`c
`potency 10r 111 u Itlng DHFR or other folate- epei . .
`enzymes, none has yet replaced MTX in the clinic. ThiS 15
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`
`Antifolates
`
`111
`
`Table 6-1. Key Features of M ethotrexate
`
`Mechanism of actio n:
`
`Metabolism:
`
`Pharmacokinetics:
`Elimination:
`Drug in teractions:
`
`Toxicity:
`
`Precautio n:
`
`Inh ibition of di hyd roto late reductase leads to partial depletio n of reduced folates.
`l'olyglutamates of methotrexate and dihydrofolate inhibit purine and thymid ylate biosynthesis.
`Converted to polyglutamates in normal and malignant tissues.
`7-H yd roxylati on in liver.
`t 11,a = 2-3 h; t 11, f3 = 8- 10 h
`Primari ly as intact drug in urine.
`l . H igh dose toxicity to normal tissues resc ue by leucovorin .
`2 . L-Asparagi nase blocks toxicity and antitumor action .
`3. Pretreatment with metho trexate increases 5- fl uorouracil and ara C nucleo tide fo rm ati on.
`4 . Nonsteroidal anti-inflammator)' age nts dec rease renal clea rance and increase toxicity.
`1. Myelos uppression
`2 . M ucositis, gastroi ntestinal epithel ial de nudation
`3. Renal tubula r o bstructi on and injury
`4 . H epatotoxicity
`5. Pneumonitis
`6 . Hypersensitivi ty
`7 . Neurotox icity
`l. Redu ce dose in proportion to dec rease in creatin ine clearance.
`2 . Do nor ad minister hi gh-dose methotrexate to patients wirh abnormal rena l timcrio n .
`3. M oni tor plasma concentrations of drug and hydrate patients during hi gh-dose therapy
`(sec Tables 6-2 and 6-4 ).
`
`. I b
`.1. .
`l tl
`f
`Prin1arily be
`c:
`.
`cause o greater ,ami 1anty wit 1 ot 1 1e cyto-
`t
`OXIc a
`· ·
`CtiVIty and host toxicity patterns of MTX and the
`C .
`lltrent I
`I
`.
`w· 1
`ac <: of evidence that these analogue compo unds
`I
`.
`.
`.
`It 1 the
`'bl
`.
`h'b·
`poss1 e excepbon of the thym1dylate synt 1ase m -
`ff'
`· 1
`·
`·
`1 I tors l1a
`d ·f·
`t·• .
`' 've cit 1er Improved therapeutic e 1cacy or a 1 -
`etent
`spectrum of clinical activity.
`
`~~~HULAR PHARMACOLOGY AND
`ANISMS OF RESISTANCE
`
`·
`In this s
`cyt
`. ectJo n the sequence of events that lead to the
`Otoxtc
`·
`·
`b
`·
`·
`With dru actton of MTX will be considered , egmnmg
`. g movement across the cell membrane, fo llowed
`by ·
`de/
`ts I ntracell ul ar metabolism to the polyglutamate
`IVative b' d'
`folat
`s,
`111 m g to d ih)rdro fo late red uctase and o ther
`' e-de
`d
`.
`and fi
`pen ent en zymes effects o n mtracellular folates,
`ll1al!y . I 'b· ·
`I
`'
`. E
`I
`f h
`' 11111 1t1 on of DNA synt 1es1s. ac 1 o
`step
`t ese
`cli1/ P1lays an essential role in determin ing the ultimate
`tea effi
`·c 1
`Jcacy and toxicity of M TX and other antu o ate
`con1
`Pounds.
`
`Transm b
`em tone Transport
`
`1'he n1
`ovement of MTX and other anti folates across
`ceiJ 1
`I
`f'
`11el11 br
`the p
`anes 1as received much attenti o n beca use o
`Opn1eotentia! role of transpo rt abno rmalities in the devel-
`nt of ·J'
`· 1
`,
`Cells b
`c Inica drug resistance (Fig. 6 -3). MTX enters
`Y an ene
`d
`· ·
`d
`rgy- ependent, temperature-sensitive, an
`conce
`.
`.
`ntt ative
`c:
`·
`.
`of spe · fi
`process that likely depends on the I ll nwon
`is anQ c tn tramembrane protein (s).34-39 T his mechanism
`Ion-de
`d
`.
`. .
`pen ent and glucose -msenstbve 4 0-43 For a
`con1p . 1
`te 1ensiv
`·
`·
`tl
`1e reade. . e rev1ew o f the cellu lar transpo rt of fo lates,
`1
`ts referred to the recent article by Antony.'~4
`
`T he n_1embrane carrier responsible fo r MTX transport in
`mammalian cells also transports the natu rally occulTing
`reduced fo lates, incl uding
`the rescue agent 5-to rmyl(cid:173)
`tetrahydroto late (leucovorin ). 38•~3-17 T hus M T X and physio(cid:173)
`logic tolates compete for cell ular entry. In additio n, tl1 ro ugh
`a process known as heteroexchange, fi·ee intracellular MTX is
`forced to efflu x fi·o m cells when high concentratio ns of
`extJ·acellular reduced folate cross tl1e cellmembrane.48
`T he influx carrier for M T X is comprised of at least two
`different systems which include ( 1) a carrier o ften referred
`to as the reduced -folate carrier with a hig her affi nity fo r
`reduced fo lates and M TX (affi nity consta nt == 0 .7 to
`6 pM) when co mpared witl1 fo lic acid (affinity consta nt ==
`2 00 pM) and (2 ) a hydrop ho bic membrane-associated
`fo late-binding p1:otein(s) (human fo late recepto r, hFR) t11at
`has a 10- to 30-fold higher afnn ity fo r folic acid and tl1e re(cid:173)
`duced folates (nanomolar ran ge ) than for MTX_3+-39,47,49-52
`MTX polyglutamates dem o nstJ·ate a 100-fold increased
`affi nity for the folate-binding protein when compared
`with tl1e mo noglutamate fo rm of MTX. 53 Whether tl1ese
`two transport systems are directly interrelated or fu nction
`separately awaits the complete mo lec ular characterization
`of each of these transpo rt systems. Recently, a process
`referred to as potocytosis, a m echanism distinct ti·o m recep(cid:173)
`to r-mediated endocytosis, was fo und to be associated with
`fo late-binding proteinss+-56 T his mechanism has bee n
`used to descri be the accumul atio n o f binding proteins in
`distinct regions o f the cell membrane known as caJJcola
`that form intracellu lar vesicles containing protein -bound
`fo lates. Presumably, folates are released tl·om their hFRs
`through changes in pH that occur within the caveola .
`O nce released fi·om the h FRs in th e caveola, the fo lates
`may then enter the intracellular space via a specifi c m em (cid:173)
`brane carrier, perhaps representing the reduced -folate car-
`
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`
`112
`
`CANCER CHEMOTHERAPY AND BIOTHERAPY
`
`Figure 6-2. Structures of tetrahydrofolate and clinically useful antifolate compounds.
`
`Pterldlne Ring
`
`p-Amlno
`Benz011te
`
`Glutemyl
`R .. ldues
`
`.~L,0~"'
`
`OCH,
`
`H~J....)
`
`N
`Trlmethoprim
`
`Cl
`
`NH,
`
`H~l N
`
`CH, -CH1
`
`Pyrimethamine
`
`1 O-Ethyl·1 O·Deaza Aminopterin
`
`CB 3717
`
`5-10 Dldeazatetrahydrofolate
`
`ZD 1694
`
`1843U89
`
`rier. Thus such a mechanism of transmembrane fc>late
`transport wou ld appear to incorporate the functions of
`both the folate- binding proteins and the reduced -folate
`carrier into one unified process.
`In the human nasopharyngeal KB carcinoma cell line
`and in monkey kidney cells, the membrane fi:.>late- binding
`protem was shown to be markedly upregu lated by fi:.>late
`depletion and, correspond ingly, downregulated in fc>late(cid:173)
`replete medium. Furthermore antibodies to hFR blocked
`transport of both folic acid and MTX, suggesti ng a role tor
`J ·
`'( 17-7 - o
`these b!Ild!Il
`·
`·
`·
`g protems 111 folate accumu at1on:"·· ·' ·'" In
`contrast, the lower-affinity red uced-folate carrier appeared
`to be responsible for the majority of fc>late transport under
`
`conditions of high, no nphysiologic extracellul ar folate
`concentratio ns. T he folate-binding proteins have been
`shown to be highly expressed in hum an ovari an carci noma
`cells, thereby providing a biochemical rationale fc>r the use
`of antifo lates that specifically employ this protein fi.x cel(cid:173)
`lular transport in the treatment of ovarian carcino ma. ' 9
`6 0
`•
`An identical folate-binding protein has been isolated from
`other human tissues, including placenta, where two dis(cid:173)
`tinct forms representing the adult and fetal forms, respec(cid:173)
`tively, have been identified(,] Usin g a monoclonal anti(cid:173)
`body to folate-binding protein, this protein was found to
`be expressed in normal ovary, lung, thyroid, kidney, and
`choroid plexus 62 ·63 T he latter locatio n suggests a possible
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`
`Antifolates
`
`113
`
`Figure 6-3. Transport systems identified far the physiologic folates and various antifolates. (Adapted from Antony et al. Blood
`79:2807, 1992.)
`
`PHYSIOLOGICAL
`SERUM FOLATE
`5-METHYLTETRAHYDROFOLATE
`
`NEWER
`ANT/FOLA TES
`
`'-·
`
`PHARMACOLOGIC FOLATES AND ANriFOLATfS
`
`I FOLIC ACID
`I
`
`5-FORMYLTETRAHYDROFOLATE METHOTREXATE (MTX)
`
`J
`(,--.-1-1,1 1 1 •
`
`l
`l '--
`' ~,..=====-.-.-~,
`l
`
`PLASMA MEMBRANE ~FOLATE RECEPTORS§ PLASMA MEMBRANE ~REDUCED FOLATE CARRIER~
`
`~PASSIVE DIFFUSION J
`
`viS
`other (?receptor-med•ated endocytoSis?)
`
`pass•ve dtHus•on
`at htgh extracellular
`. . , / ' fol ate concentr<1hon!i
`
`~....-'
`
`role for this protein in concentrating folate in the ce re(cid:173)
`brospinal fluid. Recently, a 1.1 -kilobase eDNA encod ing
`for an approximately 30-kDa folate -binding protein was
`cloned from several mammalian sources, and the gene was
`66 A proteolytic subunit
`mapped to chromosome 11q 13.64
`-
`of hFR, a soluble, hydrophilic fo late-binding protein , has
`been identified in human milk and serum. A precursor(cid:173)
`product relationship has been demonstrated by several
`laboratories for the soluble and membrane -bound t(mns
`of the folate -binding protein. 67·6H
`The exact role of these binding proteins in MTX and
`folate transport, either in trapping folatcs at the cell sur(cid:173)
`face or in their transport across the membrane, remai ns
`unclear at the present time. Transfecrion of human breast
`cancer cells that lack folate-binding protein with an h~R
`eDNA has led ro confl icting results with respect to MTX
`uptake and cytotoxicity 69 ·70 While these studies suggest a
`potential role for the folate-binding protein in tl1e trans(cid:173)
`port of physiologic folates, they do not conclusively
`demonstrate a role in MTX uptake; this result may be ex (cid:173)
`plained by the marked dependency of MTX transport on
`the red uced-folate carrier present in the specific human
`breast cancer cell line investigated. In contrast, other stud(cid:173)
`ies have shown that expression of a reduced-folate trans (cid:173)
`port protein is correlated with increased MTX transport
`and enhanced cytotoxicity in human leukemia cells. 71·72
`The accumulation of MTX in rumor cells can be influ(cid:173)
`enced by a variety of agents, including antitumor drugs
`commonly used with the anritolate in combination
`chemotherapy regimens. Vincristine increases intrace llu (cid:173)
`lar MTX by inhibitin g drug etllux. 7' ·74 The concentration
`of vincristine required to produce this eff-ect ( 10 pM),
`however, may not have direct clin ical relevance, since
`peak vincristine blood levels of only less than 0.1 p M can
`be achieved readily. In experimental chemotherapy of
`murine L 121 0 leukem ia, the seq uence of vincristine fol(cid:173)
`lowed by MTX administration did nor improve survival of
`tumor-bearing animals. For unexpl ained reasons, how (cid:173)
`ever, vincristine given 8 to 48 hours after MTX did pro-
`
`duce synergistic therapeuti c effects 74 MTX influx is in (cid:173)
`hibited by ouabain , glu cocorticoids, and cep halothin /s
`although none of these interactions has been shown to af
`feet the clinical activity of MTX. The bile salts ( cholate,
`taurocho late, and deoxycholate ) have been fo und to di (cid:173)
`minish hepatic MTX transport, and expansion of the bile
`salt pool has been considered as one potential approach
`to reduce MTX hcpatotoxicity 7 6 One of the major
`metabolites of MTX, 7 -0H -MTX, formed by the action
`of hepatic aldehyde oxidase, ~1 l so competes with MTX for
`cellular transport, thereby diminishing the intracellular
`79 The extent ro whic h this
`accumulation of MTX .77
`-
`metabolite is formed may play a role in modulating the
`cytotoxic activity of MTX, but its eff-ect on MTX trans(cid:173)
`port in the clinical setting remains uncertain.
`. The proliferative ~>r kinetic stare_ of tumor cells strongly
`mfluenccs the rate ot folate and M1 X transport. In general,
`rapidly dividing cells have a greater rate of MTX uptake and
`a decreased rare of drug efflu x when compared with cells
`that arc either in the stationary phase o r arc slowly g row(cid:173)
`ing .Ho In some experiments, indu ction of diff-erentiation of
`tumor cells, independent of eHects on growth rate,
`markedly decreased MTX transport ( 15 -fold).H' Transport
`effects, as wel l as increased requirements for DNA precur(cid:173)
`sors, increased thym idylatc synthase activity, and greater
`MTX polyglutamation all contribute to the greater cy(cid:173)
`totoxicity of MTX in rapidly dividing tumor cells. lt is
`noteworthy that the same proliteration dependency of
`MTX transport was not observed in intestinal epithel ial
`cells separated into hi gh and low proliterativc ti·actionsN2
`In addition to the hi g h -aftinity uptake syste m s de (cid:173)
`sc ribed above, a second, relatively less etl-icicnr transport
`mechanism has been descnbed ,.or drug concentrations
`in excess of 20 p i\1. si,H.' This process may represent as(cid:173)
`sivc ditfu sion and docs no r show com •)ctitio 11 be·t P
`ween
`.
`,
`t
`tolarcs and M fX or hercrocxchangc. This putar,·v .
`c cnrrv
`.
`.
`'
`process may prov1de the ration ale for the usc
`f 1 .
`·
`. .
`
`1 11g 1-
`.
`l
`.
`dosc MTX 111 c!Imca practice.
`
`0
`
`Lilly Ex. 2054
`Sandoz v. Lilly IPR2016-00318
`
`

`
`114
`
`CANCER CHEMOTHERAPY AND BIOTHERAPY
`
`The efflux ofMTX from cells takes place through mul(cid:173)
`tiple mechanisms, one of which appears to be identical to
`85 The latter accounts for greater
`the influx carrier. 42 '84 ,
`than 70% of drug efflux in Ll210 cells in the absence of
`glucose. In glucose-replete conditions, most efflux (70%)
`occurs by two other glucose- and ATP-dependent routes.
`One pathway is inhibited by bromosulfophthalein (BSP),
`probenecid, prostaglandin A, and high concentrations of
`
`KCI and is shared by cyclic nucleotides. 86- 89 Although the
`efflux pathways for MTX in human leukemic lym(cid:173)
`phoblasts (CCRF-CEM cells) are identical to those
`described for the murine cells, the BSP-sensitive route is
`quantitatively insignificantY° Further work has shown
`that the efflux pump for MTX appears to be functionally
`distinct from the P-glycoprotein-mediated, multidrug re(cid:173)
`sistance system that has been described for the transport
`of naturally occurring antineoplastic agents. 9 1
`Both in vitro and in vivo experimental systems have
`identified defective transport as a common mechanism of
`intrinsic or acquired resistance to MTX. 92
`95 However,
`-
`there are other factors, including intracellular levels of
`DHFR, affinity ofDHFRfor MTX, polyglutamation, and
`levels of thymidylate synthase (TS) that also may exert a
`strong influence on MTX cytotoxic action and whose ef(cid:173)
`fects may not be easily separable from drug transport. A
`number of MTX-resistant cell lines with functional de (cid:173)
`fects in the reduced-fo late carrier have now been de(cid:173)
`scribed, with the majority being established by single-step
`selection in the presence of MTX.96
`100 The conditions
`-
`employed to generate these mutant cell lines in vitro
`often use a higher folate concentration ( l to 5 ~1M) than
`that normally found in vivo (5 to 50 nM). Therefore, neo (cid:173)
`plastic cells with a markedly reduced capacity to transport
`folates may not be able to survive in vivo in a low-folate
`environment. 101 As an example of this, analysis of a
`murine leukemic Ll210 cell line resistant to MTX
`revealed an impaired ability to transport MTX, folic acid,
`and 5-formyltetrahydrofolate via the reduced-folate car(cid:173)
`rier system 102 However, the growth of these resistant
`cells was maintained by the use of a second transport
`system that was unable to transport MTX but whose
`activity was sufficient to allow cell growth in medium sup (cid:173)
`plemented with folate (2.2 ~1M). An IvlTX-resistant
`human lymphoblastic CCR.F-CEM/MTX cell line was
`established and maintained in physiologic concentrations
`offolate (2 nM) 103 These cells lacked the reduced -folate
`carrier protein and, solely for this reason, were resistant to
`MTX. However, these cells retained the folate -binding
`protein, and using this efficient transport process, their
`growth was maintained even in nanomolar concen (cid:173)
`trations of folic acid. This study is of particular interest
`because the concentration of folate employed was in the
`physiologic range, and thus this mechanism of transport(cid:173)
`mediated resistance may have direct clinical relevance .
`With the increasing availability of molecular reagents
`for the human folate receptor, studies have indicated that
`in selected cell lines the hFR may contribute to MTX
`transport, and changes in hFR may contribute to the
`
`development of cellular MTX resistance. Nine MT~d-
`·d ·mol
`1
`resistant clones from a human nasopharyngea ep1 er
`(KB) parent cell line that lacks the reduced-folate trans(cid:173)
`porter were established and maintained in low-fola~~
`media (1 to 10 nM). 104 Each of these sublines was MT
`resistant as a result of an impaired MTX transport that cor(cid:173)
`related with both decreased activity and decreased pro~!11
`expression of the human folate receptor. Further stu e~
`examining the molecular basis for the decreased lev~~
`hFR revealed that both the levels of hFR-specific 111 d
`and gene copy number were correspondingly decrease ·
`!o b~gin ~o identifY clinical MT_x resistanc~ on~!:~
`bas1s of nnpa1red transport, a sens1t1ve compeuuve T:X:
`placement assay using the fluorescent analogue of M
`·
`, f1 ores-
`_
`N-( 4-ammo-4-deoxy- N 10-methylpteroyl )- N-( 4 - u
`1
`ceinthiocarbamyl)-L-lysine (PT430) has been deve
`oped. 105 An analysis of 17 patients with acute lymphocyuc
`.
`ffour
`leukem1~ (ALL) revealed that blast cells from two 0 T:X:-
`pattents 111 relapse following initial treatment w1th M
`based combination chemotherapy demonstrated defec:
`tive MTX transport. While this study offers suggesuve e~
`idence that impaired transport may play an important r? ~
`in the development of clinical MTX resistance, its true 111
`cidence and clinical relevance remain to be determined.
`Significant differences in the characteristics of anuf~­
`late drug transport have prompted interest in the deve -
`·tolates
`opment of new analogues. The nonglutamated anti
`1
`such as trimetrexate and piritrexim as well as the glutamY
`ort
`,
`.
`' .
`.
`esters of MTX, do not requtre acttve cellular t1 ansp
`·
`· t nt mu-
`· ·
`d d
`an
`emonstrate acttvtty agamst transport-rests a
`. . e
`tantS. 106-108 The dibutyl ester of MTX inhibits thymtdtn
`I .
`DN
`c.om ttS
`.
`.
`.
`mcorporanon mto
`A, apparently resu ung 11
`_
`· 1 ·b·
`1o9 Tl · com
`115
`.
`.
`cc
`11111 ttory euects on thymtdme transport.
`X . mouse
`d .
`.
`1
`poun
`ts raptdly hydrolyzed to parent MT
`111
`obutY
`·
`I
`b
`P asma ut 1s converted to an alpha- or gamma-mon
`_
`derivative in human plasma; the latter is a stable come
`pound that effectively inhibits DHFR.110 The quinazohn f
`·rate 0
`1
`·fi l
`h
`] · h
`ann o ates ave a 11g er rate of influx and a owe1
`'
`.
`efflux than does MTX while aminopterin has a faster tn-
`'
`· ua-
`flux rate; these differences correlate with the greater 111
`_
`0
`cellular accumulation of the quinazolines and at11ll1.
`.
`d~!l1
`ptenn as compared with MTX after equimolar
`0
`.
`EDAM) tS
`Ill
`.
`. 11
`10-Ethyl-10-deazaaminopterin (10-
`VIVO .
`,d wtr
`·dl
`more av1 y accumulated in tumor cells compare
`1 5
`normal bone marrow or intestinal epithelium and 1a
`.
`"t)' ili~
`•
`b
`d
`roa er therapeunc activity and less marrow toXIC!
`MTX in experimental systems. 28,1 12 vVhile the parent com·
`pound (10-EDAM) has essentially no inhibitory acttYttYs
`·
`.
`d fort11
`agamst thymtdylate synthase, its polygluta111ate ·
`_
`1
`have sign ificant inhibitory activity.l 13 This effect of pol) g_
`·
`· .. 1
`ll 4 115 In con
`I
`utamatton ts sum ar to that noted for MTX.
`'
`tl ,
`·
`·
`for 1c
`trast to MTX, wh tch has a relatively poor affintty
`. d
`folate -binding proteins, several of the recently syntllestze .
`"fi I
`· 1 · ·
`-
`uch as
`anti o ate 1111tbttors of thym idylate synthase s
`. ,
`· I
`ffit11~
`CB3
`. e
`717 and ZD1694 rely heavily on the htg 1-a
`.
`b. d"
`11 6 11 7 StJlC
`fi 1
`o ate- 111 mg protems for cellular transport.
`'
`c. _
`·her 1°
`1
`r11
`t 1ese compounds are efficiently transported by eit
`e
`late transport system, they may be Jess susceptible to
`
`Lilly Ex. 2054
`Sandoz v. Lilly IPR2016-00318
`
`

`
`Antifolates
`
`115
`
`~mergence of clinical resistance resulting from alterations
`tn membrane transport. H o mofolate is a DHFR inhibitor
`that is transported primarily by the folate-binding proteins
`and has extremely potent activity against malignant cells
`overexpressing this protein. 118 This antifolate analogue
`and the thymidylate synthase inhibitors may prove to be
`particularly interesting agents for the treatment of human
`solid tumors that have developed MTX resistance due to
`etther downregulation or alterations of the reduced-folate
`carrier system . Certain of these antifolate analogue com(cid:173)
`pounds have now undergone clinical evaluation . Tri (cid:173)
`metrexate and piritrexim have completed phase I and II
`evaluation, demonstrating on ly modest activity against
`human solid tumors. 119-121 Trimetrexate combin ed with
`leucovorin has significant activity against the pulmonary
`pathogen Pneumocystis carinii, whose DHFR enzyme is
`htghly sensitive to this combination, in contrast to its
`ltmited sensitivity to the commonly used anti-infectious
`agent trimethoprim ?,l22 10-EDAM has clinical activity
`agamst a variety of human solid tumors. Its dose-limiting
`toxtctty is mucositis rather than the myelosuppression
`123 Recent
`normally associated with MTX therapy. 115
`'
`phase II testing has shown this agent to be active in
`the treatment of non-small cell lun g cancer, soft-tissue
`sarcoma , and breast cancer with overall response rates of
`17%, 14%, and 41%, respectively. J1 5,12-I,J25 While the clini(cid:173)
`cal development of CB3717 was aborted due to severe
`and unpredictable nephrotoxicity resulting fi·om precipi(cid:173)
`tatton of this compound in the nephrorubules, ZD1694,
`a more water-soluble antifo late inhibito r of thym idylate
`synthase with no associated nephrotoxicity, is presently
`Undergoi ng phase II evalu ation. 126·127
`
`Intracellular Transformation
`
`Naturally occurring fol ates exist within cells as poly(cid:173)
`g:utamates through ti1e action of the enzyme folylpoly(cid:173)
`g _utamyl synilietase (FPGS ) iliat adds up to six glutamyl
`~toups 111 gamma peptide lin kage to the folate substrate.
`f: Ius reaction serves ti1ree main purposes for folates: ( 1) it
`actlitates the accumulation ofi ntracellular folates in vast ex(cid:173)
`cess of the monoglutamate pool that is fi·eely transportable
`I
`.
`.
`Into a d
`.
`<11
`o ut of cells
`(2) It allows se ectJve mtra-
`cellu