--o
`
`Cancer Chemotherapy
`and Biological Response
`Modifiers Annual 17
`
`Edited by
`
`H.M. Pinedo
`
`Department of Medical Oncology
`University Hospital Vrije Universiteit
`Amsterdam, The Netherlands
`
`D.L. Longo
`Gerontology Research Center
`National Institutes of Health
`
`National Institute on Aging
`Baltimore, MD, U.S.A.
`
`B.A. Chnbner
`
`Division of Hematologyloncology
`Massachusetts General Hospital Cancer Center
`Boston, MA, U.S.A.
`
` 1 997
`
`Elsevier
`
`Amsterdam — Lausanne — New York - Oxford - Shannon — Sinypore - Tokyo
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`
`
`
`ELSEVIER SCIENCE B.V.
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`P.0. Box 211
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`1000 AE Amsterdam
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`The Netherlands
`
`
`ISBN 0-444-82671-8
`ISSN 0921-4410
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`© 1997 Elsevier Science B.V. All rights reserved.
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`
`Cancer Ckemarlierapy and Biological Response Modifier: Annual )7
`
`
`
`
`
`
`
`HM. Pinedo. D.L. Longo and BA. Cbabner, editors
`1997 Elsevier Science B.V.
`
`
`
`
`
`
`
`
`
`
`CHAPTER 1
`
`
`Antimetabolites
`
`
`
`
`
`
`
`
`
`
`P.G. Johnston, C.H. Takimoto, J.L. Grem, P. Fidias, M.L. Grossbard,
`
`
`
`
`
`
`B.A. Chabner, C.J. Allegra and E. Chu
`
`1.
`
`Introduction
`
`
`
`
`
`
`
`
`In the past year, significant research has been
`
`
`
`
`
`directed towards determining the mechanism of
`
`
`
`
`
`cytotoxicity for each of the antimetabolites,
`
`
`
`
`
`
`
`and the design and development of new ana-
`
`
`
`
`
`
`
`logs continues to be an exciting area. Consid-
`erable efforts have also been made to include
`
`
`
`
`
`
`
`
`
`
`
`pharmacodynamic endpoints into clinical trials
`
`
`
`
`
`to provide a more complete understanding of
`
`
`
`
`the intricacies of combination chemotherapy.
`
`
`
`
`
`
`The various studies reviewed in this year’s
`
`
`
`
`
`
`chapter provide further insight into the mech-
`anisms of action of the antimetabolites and of-
`
`
`
`
`
`
`
`
`
`
`
`
`
`fer a more detailed understanding of both the
`
`
`
`
`biochemical and molecular determinants of
`
`
`
`
`
`
`sensitivity and resistance to these agents that
`
`
`
`
`
`
`
`is required for the design of future treatment
`strategies.
`
`2. Methotrexate
`
`
`
`
`
`Methotrexate (MTX) is a tight-binding inhibi-
`
`
`
`
`
`tor of the enzyme dihydrofolate reductase
`
`
`
`
`
`(DHFR), an important enzyme involved in
`
`
`
`maintaining intracellular
`folate homeostasis.
`
`
`
`
`
`
`This enzyme catalyzes the conversion of clihy-
`drofolate
`(H2PteGlu)
`to
`tetrahydrofolate
`
`
`
`
`
`
`(H4PteGlu) with the reduced folate being a
`
`
`
`
`
`key intermediate in one-carbon transfer reac-
`
`
`
`
`
`
`
`tions. An intact enzyme function is therefore
`
`
`
`
`
`
`
`critical for the maintenance of de novo purine
`
`
`
`
`
`
`
`and thymidylate biosynthesis as well as for pro-
`
`
`
`
`
`tein synthesis and various methylation path-
`
`
`
`
`
`
`ways. As a result, DHFR represents an impor-
`
`
`
`
`
`tant target enzyme in cancer chemotherapy.
`
`
`
`2.1. Mechanism of action
`
`
`
`
`
`
`
`The precise mechanism(s) by which MTX ex-
`
`
`
`
`
`
`
`erts its cytotoxic effects remains the focus of
`
`
`
`
`considerable research efi'orts. The long-held
`
`
`
`
`
`
`view was that treatment with MTX resulted
`
`
`
`
`
`
`in depletion of the intracellular reduced folate
`
`
`
`
`
`
`
`cofactor pool via inhibition of DHFR and that
`
`
`
`
`
`depletion of these critical one-carbon donor
`substrates was then associated with inhibition
`
`
`
`
`
`
`
`
`
`
`
`of de novo purine and thymidylate biosynthe-
`
`
`
`
`
`
`
`sis. However, as has been reviewed in previous
`
`
`
`
`issues (Annuals 10-15), several
`investigators
`
`
`
`
`
`
`
`have provided evidence that the level of intra-
`
`
`
`
`
`
`
`cellular reduced folates is reduced by only 50-
`
`
`
`
`
`
`
`60% in response to MTX treatment, a level that
`
`
`
`
`
`
`would appear to be insufficient to completely
`
`
`
`
`
`
`account for the marked cytotoxic elfects of
`
`
`
`
`
`MTX. Moreover, several groups have demon-
`
`
`
`
`
`
`strated that both MTX polyglutamates and di-
`
`
`
`
`
`hydrofolate polyglutamates are able to directly
`
`
`
`
`inhibit folate-dependent enzymes other than
`
`
`
`
`DHFR including thymidylate synthase (TS),
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`
`
`2
`
`
`
`
`glycinarnide ribonucleotide (GAR) transformy-
`
`
`
`lase, and amiuoimidazolecarboxarnide ribonu-
`
`
`transformylase
`(AICAR)
`cleotide
`(Annuals
`
`
`
`
`
`10-15). Taken together,
`these studies suggest
`
`
`
`
`
`
`- that metabolic inhibition by MTX is a complex
`
`
`
`
`
`
`process mediated by both reduced folate deple-
`
`
`
`
`
`
`tion and direct inhibition of key folate-depend-
`
`
`ent enzyme pathways.
`
`
`
`
`
`
`There remains considerable debate as to the
`
`
`
`
`
`relative contribution of purine and thymidylate
`
`
`
`
`
`inhibitory efi'ects in determining the ultimate
`
`
`
`
`
`
`cytotoxicity of MTX and other antifolate ana-
`
`
`
`
`
`logs. Several studies have demonstrated that
`
`
`
`
`
`
`
`the cytotoxic activity of MTX is prevented to
`
`
`
`
`
`
`a varying degree by exogenous thymidine. In
`
`
`
`
`contrast, exogenous administration of purines
`
`
`
`
`
`
`has been shown to markedly potentiate MTX
`
`
`
`
`
`(Annuals 10-12, 16). Given these observations,
`
`
`
`
`
`
`
`it was postulated that inhibitors of DHFR that
`
`
`
`
`
`
`
`alter both de novo purine and thymidylate syn-
`
`
`
`
`
`
`
`
`thesis may be less cytotoxic than pure TS in-
`
`
`
`
`
`
`hibitors since the resulting imbalance in deox-'
`
`
`levels
`yribonuclectide triphosphate (dNTP)
`
`
`
`
`
`
`
`may be less severe. The development of new
`
`
`
`
`
`antifolate analogs with selective inhibitory ef-
`
`
`
`
`
`fects on different folate-dependent enzymes in-
`
`
`
`
`
`
`volved in purine and pyrimidine synthesis pro-
`
`
`
`
`
`
`
`vide important new tools for studying the ef-
`
`
`
`
`
`
`
`fects of MTX treatment on folate and nucleic
`
`
`
`
`
`
`acid metabolism. In this regard, Chung and
`
`
`
`
`
`
`
`Tattersall [1] investigated the effects of the de
`
`
`
`
`novo purine synthesis inhibitor 5,10-dideazate-
`
`
`
`
`trahydrofolate (DDATI-IF) on the cytotoxic
`
`
`
`
`
`
`activity of the folate-based inhibitors of TS,
`
`
`
`
`
`
`
`ICI D1694 and CB37l7. Using the murine leu-
`
`
`
`
`
`
`
`
`kemic U210 cell
`line as their model system,
`
`
`
`
`
`they observed that DDATHF,
`in a dose-de-
`
`
`
`
`
`pendent manner, reduced the cytotoxicity of
`
`
`
`
`
`
`both ICI D1694 and CB37l7. Following treat-
`
`
`
`
`
`
`
`ment with either ICI D1694 or CB37l7 a sig-
`
`
`
`
`
`
`nificant reduction in dTTP pools occurred with
`
`
`a concomitant
`time-dependent
`increase in
`
`
`
`
`
`
`
`dATP levels that reached a maximum at 12 h
`
`
`
`
`
`
`C12. 1 P. G. Johnston et al.
`
`of
`following
`treatment. Addition
`drug
`
`
`
`
`DDATHF, however, prevented the rise in
`
`
`
`
`
`
`
`
`
`dATP levels seen after ICI D1694 or CB3'7l7
`
`
`
`
`
`
`
`treatment. In fact, dATP levels were reduced to
`
`
`
`
`
`
`approximately 30% of that observed with either
`
`
`
`
`
`
`
`drug alone. Thus, the findings from this study
`
`
`
`
`
`
`provide further evidence that the imbalance in
`
`
`
`
`
`intracellular dTTP and dATP pools represents
`
`
`
`
`
`an important determinant of cytotoxicity in
`
`
`
`
`
`cells treated with antifolate compounds and
`
`
`
`
`
`provide support for the continued development
`
`
`
`
`
`
`of novel antifolate analogs that directly target
`
`
`
`
`thymidylate biosynthesis. Further studies are
`
`
`
`
`
`
`now required to determine the potential mech-
`
`
`
`
`
`
`anisms by which dNTP pool imbalance results
`in- cell death.
`
`
`
`
`2.2. Folate transport
`
`
`
`
`
`
`
`Folate transport in mammalian cells is charac-
`
`
`
`
`
`
`terized by two distinct transport systems. One
`
`
`
`
`
`
`system involves the folate receptor (FR), a
`
`
`
`
`membrane-associated, high atfinity folate bind-
`
`
`
`
`
`
`
`ing protein with alfinity constants for folic acid
`
`
`
`
`
`
`
`
`
`and reduced folates in the range of I to 50 nM.
`
`
`
`
`
`
`
`This system is a less efficient transporter of me-
`
`
`
`
`
`thotrexate (MTX) with affinity constants rang-
`
`
`
`
`
`
`
`
`ing from 0.1 to 2 p.M; however,
`it has high
`
`
`
`
`
`aflinity for certain newer synthetic antifols,
`
`
`
`
`
`
`such as ZDl694 (tomudex), DDATHF (lome-
`
`
`
`
`
`
`trexol), LY2315l4, and BWl843U89. Variable
`
`
`
`
`
`
`
`
`expression of the FR is found in a wide variety
`
`
`
`
`
`
`of normal tissues, but some malignant tumors,
`
`
`
`
`
`
`such as ovarian and cervical carcinomas ex-
`
`
`
`
`
`
`
`
`press very high levels of the FR (Annual 16).
`
`
`
`
`Structurally, the FR is a membrane-associated,
`
`
`
`
`
`
`
`
`38 kDa glycoprcte-in and it is anchored to the
`
`
`
`
`cellular membrane by a carboxyl-terminal gly-
`
`
`
`cosyl-phosphatidylinositol (GPI) tail. Protec-
`
`
`
`
`
`
`
`lytic cleavage of this GPI linkage generates a
`
`
`
`
`
`
`
`hydrophilic soluble ‘form of the FR which is
`
`
`
`
`
`
`
`present in serum and breast milk. The second
`
`
`
`
`
`folate transport system involves the reduced
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`
`
`Antimetabclites Ch. 1
`
`
`
`
`
`
`
`folate carrier (RFC) which transports reduced
`
`
`
`
`folates, e.g. 5-methyltetrahydrofolate, and anti-
`
`
`
`
`
`
`fols, e.g. methotrexate, with an aflinity constant
`
`
`
`
`
`
`
`
`ranging from 1 to 10 pM. The RFC is a much
`
`
`
`
`
`
`
`less eflicient transporter of folic acid with aifin-
`
`
`
`
`
`
`
`
`
`ity constants in the range of 200 to 400 pLM.
`
`
`
`
`
`
`Substantial progress continues to be made in
`the molecular characterization of the human
`
`
`
`
`
`
`
`
`
`
`
`
`RFC system. Over the past year, four separate
`
`laboratories
`independently isolated human
`
`
`
`
`
`
`cDNAs with high homology to one another,
`
`
`
`
`
`
`lines
`and their transfection into mutant cell
`
`
`
`
`
`completely restored reduced folate carrier ac-
`
`
`
`
`
`tivity [2—5]. These cDNAs encoded proteins
`
`
`
`
`
`
`
`with predicted molecular sizes of S8 to 68
`
`
`
`
`
`kDa, and all contained consensus sequences
`
`
`
`
`
`for N-glycosylation. Using in situ hybridiza-
`
`
`
`
`
`
`
`
`tion, it was determined that one of the RFC
`
`
`
`
`
`
`
`cDNAs mapped to a locus on the long arm
`
`
`
`
`
`
`of human chromosome 21 [4]. The functional
`
`
`
`
`
`characteristics of these cDNAs strongly suggest
`
`
`
`
`
`
`
`that they code for the human RFC transporter;
`
`
`
`
`
`
`however, additional studies to confirm their bi-
`
`
`
`
`
`ological function are in progress. Nonetheless,
`
`
`
`
`
`
`the development of these new molecular probes
`which are associated with human reduced fo-
`
`
`
`
`
`
`
`
`
`
`
`
`late and MTX transport activity should facili-
`
`
`
`
`
`
`tate our understanding of the mechanism of
`
`
`
`
`
`reduced folate transport. Moreover, the char-
`
`
`
`
`
`acterization of the expression and distribution
`
`
`
`
`
`
`of this transporter in normal and malignant
`
`
`
`
`
`
`
`
`tissues should help define the role of this gene
`
`
`
`
`in clinical MTX transport resistance.
`
`
`
`
`
`
`Recent molecular studies of the folate recep-
`
`
`
`
`
`
`tor transport system have identified at least
`
`
`
`
`
`
`three distinct FR isoforms in mammalian cells,
`
`
`
`
`
`
`
`
`and these have been referred to as FR-a (FR-
`
`
`
`
`
`
`
`
`1), FR-li (FR-2), and FR-7 [6]. The FR-cc iso-
`
`
`
`
`
`
`form was initially characterized in human na-
`
`
`
`
`
`
`sopharyngeal KB carcinoma cells and it has
`
`
`
`
`
`
`often been detected in various epithelial neo-
`
`
`
`
`
`
`
`plasms, with very high levels being present in
`
`
`
`
`
`
`most ovarian carcinomas (Annual 16). In con-
`
`3
`
`
`
`
`
`
`
`trast, the FR-B isoform was originally isolated
`
`
`
`
`
`
`
`from human placenta, and it is typically ex-
`
`
`
`
`
`
`
`pressed in low to moderate amounts in most
`
`
`
`
`
`
`normal tissues with slightly higher levels occur-
`
`
`
`
`
`ring in many non-epithelial malignant tumors.
`
`
`
`
`
`More recently, a FR-y isoforrn was character-
`
`
`
`
`
`ized in human hematopoietic cells. Because
`
`
`
`
`
`
`
`FR-qr lacked an efiicient signal for GPI modifi-
`
`
`
`
`
`cation,
`transfection and expression of FR-7
`
`
`
`
`
`
`cDNA in Chinese hamster ovary (CHO) cells
`
`
`
`
`
`
`
`resulted in poor surface expression of the FR-7
`
`
`
`
`
`protein [7]. However, because high concentra-
`
`
`
`
`
`
`
`
`tions of FR-7 were secreted into the tissue cul-
`
`
`
`
`
`
`
`ture media, the FR-7 isoforrn may represent a
`
`
`
`
`
`
`
`secreted form of the folate receptor which is
`
`
`
`
`
`
`normally found at low concentrations in serum
`
`
`
`
`
`
`but in dramatically increased levels in folate
`
`
`
`
`
`
`deficient states. Like the other isoforrns, FR-1r
`
`
`
`
`
`
`
`has a high afiinity for folic acid; however, it
`
`
`
`
`does not display a stereospecific preference
`
`
`
`
`
`
`
`
`for binding to reduced folates as do the FR-oz
`
`
`
`and FR-[3 isoforms [7].
`
`
`
`
`
`
`The regulation of FR-cc gene expression was
`studied in five different ovarian cancer cell lines
`
`
`
`
`
`
`
`
`
`
`
`
`
`following growth in tissue culture media con-
`
`
`
`
`
`
`taining high and low folate concentrations [8].
`
`
`
`
`
`
`
`
`
`In four of five cell lines, stable changes in the
`
`
`
`
`
`
`expression of FR-or were not observed when
`the extracellular folate concentration was low-
`
`
`
`
`
`
`
`
`
`
`
`
`
`ered to physiologic levels (2 nM). Only in one
`
`
`
`
`
`
`cell line (SKOV3) was there a persistent stable
`
`
`
`
`
`
`2-fold increase in FR-or protein expression and
`
`
`
`
`
`
`this corresponded to a 1.5-fold increase in FR-
`oc mRNA levels. These authors concluded that
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`in vitro over expression of the FR-ca isoforrn in
`
`
`
`
`
`
`
`ovarian cancer cell lines was generally not sen-
`sitive to extracellular folate concentrations. In
`
`
`
`
`
`
`
`
`
`
`
`another series of experiments, Orr and col-
`
`
`
`
`
`leagues examined the regulation and expression
`
`
`
`
`
`
`
`of FR-cc isoforrn in the human squamous cell
`
`
`
`
`carcinoma cell
`line, UMSCC38, which ex-
`
`
`
`
`
`
`
`
`presses four to six copies of the FR-cl gene
`
`
`
`
`
`
`
`[9]. Despite the amplification of the gene, these
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`
`4
`
`
`
`
`
`
`
`cells did not overexpress FR-ct protein. Further
`
`molecular analysis
`identified three distinct
`
`
`
`
`
`
`
`
`point mutants of the FR-oz gene in these cells.
`
`
`
`
`Surprisingly,
`transfection and expression of
`
`
`
`
`
`these mutant FR-cc cDNAs into wild-type
`
`
`
`
`
`
`
`MAIO4 monkey kidney cells resulted in de-
`
`
`
`creased endogenous folate receptor activity
`
`
`
`consistent with a dominant-negative phenotype
`
`
`
`
`
`
`
`[9]. The mechanism by which these human FR-
`
`
`
`
`
`
`
`oz point mutants reduced the activity of the
`
`
`
`
`
`
`normal MAIO4 folate receptor was not charac-
`
`
`
`
`
`terized; however, the authors hypothesized that
`
`
`
`
`
`aggregation between mutant and normal FR—ol
`
`
`
`
`
`
`may have interfered with normal folate binding
`
`
`
`
`
`and transport. Clearly, additional studies are
`
`
`
`
`
`
`needed to test this hypothesis. Finally, exposing
`
`
`
`
`
`UMSCC38 cells to differentiating agents such
`
`
`
`
`
`as hydrocortiscne increased the expression of
`
`
`
`
`
`
`
`
`FR-a protein on the cell surface by 8-fold as
`
`
`
`
`
`measured by Western immunoblots [10]. This
`
`
`
`
`finding suggests a potentially useful clinical
`
`
`
`
`method for enhancing receptor-mediated folate
`
`
`
`
`transport in cancer chemotherapy [1 1].
`
`
`
`
`
`Two different pathways have been proposed
`
`
`
`
`
`
`for the mechanism of FR-mediated folate up-
`
`
`
`
`
`take. One is classic receptor-mediated endocy-
`
`
`
`
`
`tosis where the folate ligand-receptor complex
`
`
`
`
`
`
`is internalized at c1athrin—coated pits to ulti-
`
`
`
`
`
`mately form secondary lysoscmes within the
`
`
`
`
`
`
`
`cell. A second more novel mechanism of small _
`
`
`
`
`
`molecule uptake, termed potocytosis, was orig-
`
`
`
`
`
`
`inally described for the folate receptor (Annual
`
`
`
`
`
`15). In potocytosis, the folate ligand—receptor
`
`
`
`
`complexes cluster within specific invaginations
`
`
`
`
`
`
`
`on the cell surface, called caveolae, which in-
`
`
`
`
`
`
`
`
`ternalize by budding cfi‘ within the cell to form
`
`
`
`
`
`
`specific vesicles. Lowering of the pH within
`
`
`
`
`
`
`these vesicles causes the folate to dissociate
`
`
`
`
`
`
`
`
`from the receptor where it can then be trans-
`located across the cell membrane via a second
`
`
`
`
`
`
`
`
`
`
`
`transport system, possibly the reduced folate
`
`
`
`
`
`
`
`carrier. As detailed in last year’s Annual, the
`
`
`
`
`
`mechanism of potocytosis was questioned be-
`
`
`Ch. I P.G. Johnston er al.
`
`
`
`
`
`
`
`
`
`
`
`cause of recent evidence that clustering of the
`
`
`
`
`
`
`
`folate receptor within caveolae on the cell sur-
`
`
`
`
`
`
`
`face was the result of a technical artifact (An-
`
`
`
`
`
`
`
`nual l6). However, Ritter et 21!. recently found
`no role for the involvement of clathrin-coated
`
`
`
`
`
`
`
`
`
`
`
`
`pits in the receptor-mediated uptake of folate
`
`
`
`
`
`
`
`into mutant cells [12]. Folate uptake was not
`
`
`
`
`
`
`enhanced even when the normal GPI mem-
`
`
`
`
`
`
`
`brane anchor of the folate receptor was re-
`
`
`
`
`
`placed with a low density lipoprotein receptor
`
`
`
`
`transmembrane domain which promoted recep-
`
`
`
`
`
`tor localization within the clathrin-coated pits.
`
`
`
`
`
`
`Furthermore, Smart et al. reported that activa-
`
`
`
`
`
`
`
`
`tors of protein kinase C did not inhibit endo-
`
`
`
`
`
`
`
`cytosis, but were potent inhibitors of folate up-
`
`
`
`
`
`take and potocytosis, again supporting the
`
`
`
`
`
`
`view that these pathways were functionally dis-
`
`
`
`
`
`
`
`
`tinct [13]. Both of these reports tend to support
`
`
`
`
`potocytosis being a mechanism of FR-mediated
`
`
`
`
`
`
`
`uptake. In contrast, a study by Rijnboutt et al.
`
`
`using immunoelectron microscopy, demon-
`
`
`
`
`
`
`strated the presence cf folate receptors along
`
`
`
`
`
`
`
`the entire endocytic pathway in KB cells. The
`
`
`
`
`
`receptor was present in clathrin-coated buds,
`
`
`
`
`
`vesicles, and in endcscmal vacuoles,
`leading
`
`
`
`
`
`
`
`these authors to conclude that most,
`if not
`
`
`
`
`
`
`
`all, of the folate receptors bypass the caveolae,
`
`
`
`
`
`and instead utilize the traditional endocytosis
`
`
`
`
`
`pathway [14]. Likewise, several other laborato-
`
`
`
`
`
`
`
`ries [l5—l7} found no evidence for coupling of
`
`
`
`
`
`the RFC system with folate receptor—mediated
`
`
`
`
`
`
`uptake as suggested by the potocytosis hypoth-
`
`
`
`
`
`
`
`esis. A recent report that may reconcile these
`
`
`
`
`
`two differing viewpoints comesfrom fluores-
`
`
`
`
`cence microscopy experiments conducted by
`
`
`
`
`
`Turek et al.
`[[8]. Proteins covalently conju-
`
`
`
`
`
`
`
`
`gated to folic acid were internalized by KB cells
`
`after binding to surface
`folate receptors.
`
`
`
`
`Although the ligand-folate receptor complexes
`
`
`
`
`
`
`were initially clustered within the uncoated cav-
`
`
`
`
`
`
`
`
`
`eolae on the cell surface, by 15 min, the cav-
`
`
`
`
`eolae-mediated pathway converged with the
`
`
`
`
`
`
`clathrin-coated pit pathway. Thus, in the KB
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`
`Antimetabolites Ch. 1
`
`
`
`
`
`
`
`
`cell line, potocytosis and classic receptor medi-
`
`
`
`
`
`
`ated endocytosis may be directly related. At
`
`
`
`
`
`
`present, the precise mechanism(s) by which fo-
`
`
`
`
`
`late receptor-mediated uptake is mediated re-
`
`
`
`
`
`
`mains uncertain, but clearly this remains an
`
`
`
`area of active investigation.
`
`
`
`
`
`Several laboratories have begun to determine
`
`
`
`
`
`
`
`the relative importance of the FR and RFC
`
`
`
`
`
`
`transport systems in the uptake of antifolates
`
`
`
`
`
`in clinical cancer chemotherapy. Westerhof and
`
`
`
`
`
`colleagues compared the transport and cytotox-
`
`
`
`
`
`
`
`icity of a series of different antifolates in cells
`
`
`
`
`
`which simultaneously express both the RFC
`
`
`
`
`
`
`
`and the FR (KB, MAIO4 and IGROVI cells)
`
`
`
`
`
`
`[16]. The drugs analyzed included MTX and
`
`
`
`
`
`
`10-EdAM, relatively specific substrates for the
`
`
`
`
`
`
`RFC; DDATHF and ZDl694, substrates for
`
`
`
`
`
`
`
`both the RFC and the folate receptor; and
`
`
`
`
`
`
`CB37l7, a specific substrate for the folate re-
`
`
`
`
`
`
`ceptor only. Folate transport was examined in
`
`
`
`
`
`
`
`
`both high (2.2 1.1M folic acid) and low folate-
`
`
`
`
`
`containing (1-10 nM leucovorin) media.
`In
`
`
`
`
`
`
`each cell line tested, drug sensitivity correlated
`
`
`
`
`
`
`
`best with RFC expression and not with FR
`
`
`expression. Furthermore, growth inhibition
`
`
`
`
`
`
`was blocked much more efliciently by leucovor-
`
`
`
`
`
`
`
`in, which competes for RFC uptake, and not
`
`
`
`
`
`
`
`by folic acid, which preferentially binds to the
`
`
`
`
`
`folate receptor. These authors concluded that
`
`
`
`
`
`
`
`the RFC was the preferential route of entry
`
`
`
`
`
`
`for antifolate compounds even in cells express-
`
`
`
`
`
`
`
`
`
`ing high levels of FR. Only at very low (nano-
`
`
`
`
`molar) extracellular concentrations of reduced
`
`
`
`
`
`
`
`folatcs did the folate receptor contribute to the
`
`
`
`
`
`
`
`
`uptake of folate into cells. Schultz et al. also
`
`
`
`
`
`
`reported a lack of correlation between FR ex-
`
`
`
`
`
`
`pression and DDATHF sensitivity in cell lines,
`
`
`
`
`
`
`
`such as KB and IGROVI, which express high
`
`
`
`
`
`
`
`levels of FR [19]. Thus, the increased expres-
`
`
`
`
`
`
`
`
`
`sion of FR in some tumors may not be easily
`
`
`
`
`
`
`exploited as a means of increasing the thera-
`
`
`
`
`
`peutic efficacy of antifolate chemotherapy as
`
`
`was originally proposed.
`
`5
`
`
`
`
`
`
`A different experimental approach was taken
`
`
`
`
`
`
`
`by Spinella and colleagues in their analysis of
`
`
`
`
`
`
`MTX uptake in two diiferent murine leukemia
`
`
`
`
`
`
`
`
`
`Ll2l0 cell lines. One cell line was wild-type and
`
`
`
`
`
`
`
`
`expressed the RFC while the second one was a
`
`
`
`
`
`
`mutant cell line lacking a functional RFC but
`
`
`
`
`
`
`
`transfected with the FR cDNA [15]. The FR-
`
`
`
`
`expressing cells effectively transported reduced
`
`
`
`
`
`
`folatcs and folic acid at physiologically relevant
`
`
`
`
`
`
`
`low concentrations of 10-50 nM and MTX, at
`
`
`
`
`
`
`concentrations of 0.1 pLM. At higher concentra-
`
`
`
`
`tions, however, FR-mediated uptake appeared
`
`
`
`
`
`
`to play a relatively minor role compared to
`
`
`
`RFC-mediated uptake. Furthermore, transport
`
`
`
`
`
`
`
`
`via the FR was blocked by the presence of
`
`
`
`
`
`
`physiologic concentrations of folic acid of 10
`
`
`
`
`
`nM. Together, these experiments provide fur-
`
`
`
`
`
`
`
`
`
`ther support to the view that the RFC system is
`
`
`
`
`the more pharmacologically relevant transport-
`er of MTX in mammalian cells.
`
`
`
`
`
`Previous issues of this Annual described new
`
`
`
`
`
`
`
`
`
`
`
`experimental strategies to target tumors with
`
`
`
`
`
`
`
`high surface expression of FR by using folic
`
`
`
`
`acid conjugated to difi'erent
`toxins (Annual
`
`
`
`
`
`
`
`16). As another example of this therapeutic ap-
`
`
`
`
`
`proach, Kranz and colleagues designed conju-
`
`
`
`
`
`
`
`gates of folic acid and anti-T-cell receptor anti-
`
`
`
`
`
`
`bodies to target mouse tumor cells expressing
`
`
`
`
`
`
`
`high levels of the folate receptor [20]. Binding
`
`
`
`
`
`
`
`of the folate—antibody complex to the cell sur-
`
`
`
`
`
`face stimulated a cytotoxic T-cell response in
`
`
`
`
`
`
`
`vitro resulting in tumor lysis. Thus, the folate
`
`
`
`
`
`
`receptor may represent an attractive target for
`
`
`
`novel tumor-specific antigen-directed immuno-
`
`logic therapies.
`
`2.3.
`
`Intracellular metabolism
`
`
`The intracellular metabolism of MTX and oth-
`
`
`
`
`
`
`
`
`
`
`
`er antifolates to their polyglutamate metabolite
`
`
`
`
`
`
`
`forms by the enzyme FPGS plays a critical role
`
`
`
`
`
`
`in determining their cytotoxic action and ther-
`
`
`
`
`
`apeutic selectivity. Previous studies have dem-
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`Lilly Ex. 2074
`Sandoz V. Lilly IPR20l6-00318
`
`Lilly Ex. 2074
`Sandoz v. Lilly IPR2016-00318
`
`

`
`f.
`
`6
`
`
`
`
`
`onstrated that polyglutamate metabolites are
`
`
`
`
`
`selectively retained within the cell prolonging
`
`
`
`
`
`their intracellular half-life. In addition, MTX
`
`
`
`polyglutamates and antifolate polyglutamates
`
`
`
`
`
`
`
`
`have been shown to be more effective and po-
`
`
`
`
`
`tent inhibitors of various folate-dependent en-
`
`
`
`
`
`
`zymes such as TS, AICAR transformylase, and
`
`
`
`
`GAR transformylase (Annuals 10-16). Signifi-
`
`
`
`
`
`
`cant research efforts have focused on FPGS
`
`
`
`
`
`
`given its role in the sequential gamma-addition
`
`
`
`
`
`
`
`of glutamate to the terminal glutamyl moiety of
`
`
`
`
`
`
`folates and antifolates, and human FPGS has
`
`
`
`
`
`
`been recently cloned and expressed in a mam-
`
`
`
`
`
`
`
`malian cell deficient in FPGS. In contrast to
`
`
`
`
`
`the process of polyglutamation, the hydrolytic
`
`
`
`
`
`cleavage of folyl and antifolylpolyglutamates as
`
`
`
`
`catalyzed by gamma-glutamyl hydrolase (GH),
`
`
`
`
`
`
`
`an enzyme whose cellular form is primarily ly-
`
`
`
`
`
`
`
`sosomal in origin, has been relatively less well-
`
`
`
`
`
`
`studied. Although GH has been partially puri-
`
`
`
`
`
`
`fied and investigated from various sources, its
`
`
`
`
`
`role in mediating cellular polyglutamate forma-
`
`
`
`
`
`
`
`
`tion is just beginning to be established. To ad-
`
`
`
`
`
`
`
`
`dress this issue, Yao et al. [21] examined the
`
`
`
`
`
`
`
`
`effects of GH on the formation of folyl and
`
`
`
`
`antifolylpolyglutamates in cultured rat hepato-
`
`
`
`
`
`
`
`
`ma H35 cells. In addition to the parental H35
`
`
`
`
`
`
`
`cell
`line,
`they studied a subline of H35 cells
`
`
`
`
`
`
`
`(H35D) that had been made resistant to the
`
`
`
`antifolate analog 5,10-dideazatetrahydrofolate
`
`
`
`
`
`(DDATHF). Previously, they had reported that
`the H35D cell line exhibited a 7-fold increase in
`
`
`
`
`
`
`
`
`
`
`
`
`
`GH enzyme activity compared to parent H35
`
`
`
`
`
`
`
`
`cells with no difference in the level of FPGS
`
`
`
`
`
`
`
`activity (Aimual 15). In the present study, they
`
`
`
`
`
`
`demonstrated that the hydrolysis rate of MTX
`
`
`
`
`polyglutamates with isolated, intact lysosomes
`
`
`
`
`
`
`
`
`was 4—5-fold greater in H35D cells than in pa-
`
`
`
`
`
`
`
`
`rent H35 cells. In contrast to MTX, the total
`
`
`
`
`intracellular folate concentration was nearly
`
`
`
`
`
`
`
`identical in both H35 and H35D cells following
`
`
`
`
`
`
`
`exposure to folic acid up to concentrations of
`
`
`
`
`
`
`
`10 ,uM. However, the chain length of folylpo-
`
`Ch. 1 P. G. Johnston et al.
`
`
`
`
`
`
`
`
`
`
`
`lyglutamates was mainly tri- and tetragluta-
`
`
`
`
`
`
`
`
`mates in H3SD cells unlike the parent H35 cells
`
`
`
`
`
`where penta- and hexaglutamate forms predo-
`
`
`
`
`
`
`minated. At higher concentrations of folic acid
`
`
`
`
`
`
`
`(50 and 100 1.1M), the totalfolate accumulation
`
`
`
`
`
`
`
`in H35D cells was significantly less than that
`
`
`
`
`
`
`
`observed in H35 cells. The lower chain polyglu-
`
`
`
`
`
`
`tamate forms were again the principal species
`
`
`
`
`
`
`measured in the resistant H35D cells. Thus,
`
`
`
`
`
`
`
`this study provides evidence for the role of
`
`
`
`
`
`GH in mediating polyglutamate formation for
`
`
`
`
`
`both naturally occurring folates and antifo-
`
`
`
`
`
`lates. Further investigations into the regulation
`
`
`
`
`
`
`of this lysosomal enzyme should provide in-
`
`
`
`
`
`
`
`
`
`sights that may serve as the basis for the design
`
`
`
`
`and development of new therapeutic ap-
`
`
`
`
`
`
`proaches that can either circumvent andfor pre-
`
`
`
`
`
`
`vent the development of antifolate drug resist-
`ance.
`
`
`
`
`
`
`Previous studies identified the prognostic sig-
`
`
`
`
`
`nifieance of cytogenetic abnormalities in child-
`
`
`
`
`
`hood ALL with hyperdiploidy being a good
`
`
`
`
`
`prognostic feature. Subsequent work by White-
`
`
`
`
`
`
`head and his colleagues (Annual 15) demon-
`
`
`
`
`
`
`strated that patients with ALL and hyperdi-
`
`
`
`
`ploid lymphoblasts synthesized higher
`levels
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`of MTX polyglutamates than those patients
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`whose lymphoblasts were either aneuploid or
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`diploid in nature. In addition, children with
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`B-cell ALL whose Iymphoblasts accumulated
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`higher levels of MTX polyglutamates in vitro
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`experienced improved 5-year
`survival
`rates
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`compared to those children whose ALL blasts
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`generated lower levels of MTX polyglutamates.
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`While MTX has played an integral role in
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`the treatment of ALL,
`the optimal dose of
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`MTX has remained the subject of much con-
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`troversy. Although one randomized study had
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`shown that treatment with high-dose MTX was
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`associated with significantly improved event-
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`free survival compared to low-dose MTX,
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`many questions have been raised about the ra-
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`tionale for high-dose MTX. In particular, it has
`
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`
`Lilly Ex. 2074
`Sandoz V. Lilly IPR20l6-00318
`
`Lilly Ex. 2074
`Sandoz v. Lilly IPR2016-00318
`
`

`
`Antimetabolites Ch. I
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`been suggested that higher extracellular MTX
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`concentrations may not necessarily produce
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`higher
`intracellular concentrations in ALL
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`blasts given the potential for saturation of re-
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`ceptor-mediated MTX uptake and saturation
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`of metabolism to the active MTX polygluta-
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`mate forms. To address this issue, Synold et
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`al. [22] compared the formation of MTX poly-
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`glutamate formation in bone marrow blasts in
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`patients treated with either low-dose (30 mglmz
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`p.o. every 6 h for 6 doses) or high—dose (1 g/m2
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`given i.v. over 24 h) MTX. They observed that
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`high-dose MTX resulted in significantly higher
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`concentrations of MTX polyglutamates in leu-
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`kemic blasts cells in vivo when compared to
`
`achieved by oral,
`low-dose MTX.
`those
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`Although both regimens were able to maintain
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`plasma MTX concentrations above 0.1 _uM for
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`a similar period of time (44 h), the high-dose
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`MTX regimen achieved significantly higher
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`MTX plasma concentrations (12-fold)
`than
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`the low-dose regimen. Moreover, the high—dose
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`regimen produced markedly higher concentra-
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`tions of long-chain MTX polyglutamates (4-6
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`glutamyl residues) when compared to the low-
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`dose schedule. They also noted that T-cell
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`blasts accumulated much lower MTX polyglu—
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`tamates than B-cell blasts, and as well,
`they
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`found that nomhyperdiploid B-cell blasts gen-
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`erated far lower levels of MTX polyglutamates
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`than hyperdiploid B—cells bl