EXHIBIT 2018
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`EXHIBIT 2017
`
`Cephalon Exhibit 2017
`Fresenius v. Cephalon
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`|PR2016-00111
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`7.11abner,
`Director of Clinical Research
`Massachusetts General Hospital Cancer Center
`Professor of Medicine
`Harvard Medical School
`Boston, Massachusetts
`
`Ban .-72111
`Division of Hematology
`Brigham and Women's Hospital
`Deputy Editor
`New England Journal of Medicine
`Boston, Massachusetts
`
`trkt Wolters Kluwer I Lippincott Williams & Wilkins
`
`Health
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`CEPHALON, INC. -- EXHIBIT 2017 0001
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`Library of Congress Cataloging-in-Publication Data
`Cancer chemotherapy and hiotherapy : principles and practice / editors, Bruce A. Chabner, Dan L. Longo. —5th ed.
`p.; CM.
`Includes bibliographical references and index.
`Summary: "Updated to include the newest drugs and those currently in development, Cancer Chemotherapy and
`Biotherapy, Fifth Edition is a comprehensive reference on the preclinical and clinical pharmacology of anticancer agents.
`Organized by drug class, the book provides the latest information on all drugs and biological agents—their mechanisms
`of action, interactions with other agents, toxicities, side effects, and mechanisms of resistance. Chapters emphasize
`pharmacology and mechanisms of action at the molecular and cellular levels, followed by clinical activity and toxicity,
`both acute and delayed The authors explain the rationale for use of drugs in specific schedules and combinations and offer
`guidelines for dose adjustment in particular situations. The previous edition was one of"Doody's CoreTitles 2009." This
`edition's introduction includes timely information on general strategies for drug usage, the science of drug discovery
`and development, economic and regulatory aspects of cancer drug development, and principles of pharmacokinetics.
`Eight new chapters have been added and more than twenty have been significantly revised" Provided by publisher.
`ISBN 978-1-60547-431-1 (hardback : alk. paper)
`1. Cancer --Chemotherapy. 2. Cancer--Immunotherapy, 3. Antineoplastic agents. 4. Biological response modifiers.
`I. Chabner, Bruce. II. Longo, Dan L. (Dan Louis), 1949-
`IDNLM: 1. Neoplasms—drug therapy. 2. Antineoplastic Agents—therapeutic use. 3. Biological Products—
`therapeutic use. QZ 267 C21515 20111
`
`RC271.C5C32219 7011
`616.99'4-061—dc22
`
`2010023843
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`Care has been taken to confirm the accuracy of the information presented and to describe generally accepted practices.
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`remains the professional responsibility of the practitioner.
`The authors, editors, and publisher have exerted every efkrt to ensure that drug selection and dosage set forth ill
`this text are in accordance with current recommendations and practice at the time of publication. I however, in view
`of ongoing research, changes in government regulations, and the constant flow of information 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.TEis is particularly important when the recommended agent is a new
`or infrequently employed drug.
`Some drugs and medical devices presented in the publication have Food and Drug Administration (FDA) clearance
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`CEPHALON, INC. -- EXHIBIT 2017 0002
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`(cid:9)
`

`
`CHAPTER
`
`Alkylating Agents
`
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`Stanton L. Gerson, Ahna D. Bulgar, Lachelle D. Weeks, and Bruce A. Chabner
`
`The alkylating agents arc antitumor drugs that act through the cova-
`lent binding of alkyl groups to cellular molecules. This binding is
`mediated by reactive intermediates formed from a more parent
`alkylating compound. Historically, the alkylating agents have played
`an important role in the development of cancer chemotherapy.
`The nitrogen mustards mechlorethamine (HN„ "nitrogen mus-
`tard") and tris(f3-chloroethyl)amine (HN3) were the first nonhor-
`monal agents to show significant antitumor activity in humans.'''
`The clinical trials of nitrogen mustards in patients with lymphomas
`evolved from the observation that lymphoid atrophy, in addition
`to lung and mucous membrane irritation, was produced by sulfur
`mustard during World War I. Antitumor evaluation' showed that the
`related but less reactive nitrogen mustards, the bischloroethylam-
`ines (Fig. 14A-1), were less toxic and caused regression of lymphoid
`tumors in mice. The first clinical studies produced dramatic tumor
`regressions in some patients with lymphoma, and the antitumor
`effects were confirmed by an organized multi-institution study.1 '2
`This demonstration of efficacy encouraged further efforts to find
`chemical agents with antitumor activity, leading to the wide vari-
`ety of antitumor agents in use today. Nonclassical alkylating agents
`include methylating agents such as procarbazine and temozolomide
`and are discussed later in this chapter. Alkylating agents, despite the
`
`CI — CH2CH2
`
`CI — CH2CH2
`
`S
`
`A
`Bischloroethylsulfide (sulfur mustard).
`
`CI — CH2CH2
`
`CI —
`
`N — R
`
`B
`Bischloroethylamine (nitrogen mustard general
`structure). —R = —CH3 in mechlorethamine.—R = —CH2CH2CI
`in tris((3-chloroethyl)amine.
`
`I (;111(1, '',11.3:2-'"'j (cid:9)
`Structures of bischloroethylsulfide and bischloroethylam-
`ina A. Bischloroethylsulfide (sulfur mustard). B. Bischloroethylamine (nitrogen mus-
`tard general structure).
`
`enthusiastic development of targeted agents, continue to occupy a
`central position in cancer chemotherapy, both in conventional com-
`bination regimens and in high-dose protocols with hematopoietic
`cell transplantation (PICT). Because of their linear dose-response
`relationship in cell culture experiments,'' these drugs have become
`primary tools used in FICT for a variety of diseases. Better appreci-
`ation of resistance mechanisms and development of targeting agents
`to block these resistance pathways promise to improve the efficacy
`of alkylating agents.
`
`Alkylating Reactions
`
`An alkylation reaction can occur by two mechanisms: SN1 and SN2.
`In SN I reactions, the rate-limiting step is the formation of a carbo-
`nium ion that can react rapidly with a nucleophile. This reaction
`follows first-order kinetics with a rate that depends solely on the
`concentration of the alkylating agent. In contrast, SN2 reactions
`follow second-order kinetics and depend on the concentrations of
`both the alkylating agent and the nucleophile. Such reactions involve
`a transition-state entity formed by both reactants that decomposes
`to form the alkylated cellular constituent. Agents such as chloroeth-
`ylnitrosoureas, through a SN 1-type of mechanism, can form cova-
`lent adducts with oxygen and nitrogen atoms in DNA. Compounds
`with SN2 predominant mechanisms, such as busulfan, tend to react
`more slowly, with little alkylation of oxygen sites. Because alky-
`lating agents are designed to produce reactive intermediates, the
`parent compounds typically have short elimination half-lives of less
`than 5 hours.
`As a class, the alkylating agents share a common target (DNA)
`and are cytotoxic, mutagenic, and carcinogenic.The activity of most
`alkylating agents is enhanced by radiation, hyperthermia, nitroimi-
`dazoles, glutathione depletion, and inhibition of DNA repair. They
`differ greatly, however, in their toxicity profiles and antitumor
`activity. These differences arc undoubtedly the result of differences
`in pharmacokinctic features, lipid solubility, ability to penetrate the
`central nervous system (CNS), membrane transport properties,
`detoxification reactions, and specific enzymatic reactions capable
`of repairing alkylation sites on DNA.' -7 Application of techniques
`such as magnetic resonance imaging and mass spectrometry to the
`
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`267
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`CEPHALON, INC. -- EXHIBIT 2017 0003
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`Chapter 14A 0 Classical Alkylating Agents
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`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`
`CH3
`CI-CH2CH2-N-CH2CH2CI
`
`CH3
`.3
`CI-CH2CH2-N+
`
`CH3 (cid:9)
`
`H
`
`CI-CH2CH2-N-CH2CH2-N-R1
`
`RrNH2
`
`R2NH2 (cid:9)
`
`H (cid:9)
`
`CH3
`
`R2N-CH2CH2-N-CH2CH2-NR1
`
`Alkylation mechanism of nitrogen mustards. (From Colvin
`M. Molecular pharmacology of alkylating agents. In: Cooke ST, Prestayko AW.
`Cancer and Chemotherapy, vol 3. New York: Academic Press, 1981:291.)
`
`study of the alkylation mechanism and the chemical nature of the
`intermediates involved have led to a detailed understanding of these
`reactions." Such approaches, coupled with improved techniques
`for studying cellular damage'" and for determining mechanisms
`of detoxification,' make it possible to predict sites of alkylation of
`an agent and allow scientists to understand and modify the biologic
`consequences of such alkylations.
`
`AlIcylating Agents Used Clinically
`
`The important pharmacologic properties of the clinically useful
`alkylating agents are summarized in Table 14A-1.
`
`Llitrogen Mustards
`
`The prototypic alkylating agents have been the bischloroethylam-
`ines or nitrogen mustards. The first nitrogen mustard to be used
`extensively in the clinic was mechlorethamine (mustine) (Fig. 14A-1),
`sometimes referred to by its original code name I-IN2 or by the
`term nitrogen mustard. The mechanism of alkylation by the nitrogen
`mustards is shown in Figure 14A-2. In the initial step, chlorine is
`lost and the a-carbon reacts with the nucleophilic nitrogen atom to
`form the cyclic, positively charged, and very reactive aziridinium
`moiety. Reaction of the aziridinium ring with a nucleophile (elec-
`tron-rich atom) yields the initial alkylated product. Formation of a
`
`Alkylating Reactions (cid:9)
`
`269
`
`second aziridinium by the remaining chloroethyl group allows for
`a second alkylation, which produces a cross-link between the two
`alkylated nucleophiles.
`Numerous analogs of mechlorethamine were synthesized in
`which the methyl group was replaced by a variety of chemical
`groups that stabilized the molecule. Most of these compounds
`proved to have less antitumor activity than mechlorethamine, but
`many other derivatives have a higher therapeutic index, a broader
`range of clinical activity, and can be administered both orally and
`intravenously. These drugs, which for the most part have replaced
`mechlorethamine in clinical use, are melphalan (L-phenylalanine
`mustard), chlorambucil, bendamustine, cyclophosphamide, and ifosfamide
`(Fig. 14A-3). The latter two agents are unique in that they require
`metabolic activation and undergo a complex series of activation
`and degradation reactions (to be described in detail later in this
`chapter).
`These derivatives have electron-rich groups substituted on the
`nitrogen atom. This alteration reduces the electrophilicity of the
`nitrogen and renders the molecules less reactive. Melphalan and
`chlorambucil retain alkylating activities and seem to be more tumor
`selective than nitrogen mustard. Cyclophosphamide and ifosfamide,
`on the other hand, possess no intrinsic alkylating activity and must
`be metabolized to produce alkylating compounds.
`Cyclophosphamide remains the most widely used alkylat-
`ing agent." It is an essential component of drug regimens for
`non-Hodgkin's lymphoma (NHL) (CHOP—cyclophosphamide,
`doxorubicin, vincristine (oncovin), prednisone), other lymphoid
`malignancies, and solid tumors in children. Additionally, it is used
`in combination treatments for breast cancer, and in high-dose che-
`motherapy with bone marrow restoration.
`Ifosfamide, an isomeric analog of cyclophosphamide, was intro-
`duced into clinical use in 1972. It is currently approved for the
`treatment of relapsed testicular germ cell tumors" and for the
`treatment of both pediatric and adult soft tissue sarcomas.15'16 It is
`used in combination with etoposide and carboplatin for relapsed
`lymphomas.
`Melphalan is primarily employed in multiple myeloma," occa-
`sionally in malignant melanoma and in high-dose chemotherapy
`with marrow transplantation.
`
`CI
`
`CI
`NN
`N
`AV
`CI
`
`CH2CHCO2H
`
`NH2
`
`CI
`NN.
`
`Cl '
`
`CH2CH2CH2CO2H
`
`
`
`CI
`
`Melphalan
`
`Chlorambucil
`
`OH
`
`N
`
`L.H3
`
`Bendamustine
`
`CI
`
`H
`CIS (cid:9)
`N
`N—P (
`0 (cid:9) /
`
`CI (cid:9)
`
`Cyclophosphamide
`
`I:1(m RI: (cid:9)
`
`Alkylating agent structures.
`
`0
`
`CI (cid:9)
`NN
`N—P (
`H
`
`CI (cid:9)
`
`II
`DN-
`P-N
`
`LA
`
`Ifosfamide (cid:9)
`
`Thio-TEPA
`
`att'- (cid:9)
`
`E'3
`
`Ls.. -
`
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`(cid:9)
`(cid:9)
`

`
`270 (cid:9)
`
`Chapter 14A o Classical Alkylating Agents
`
`Chlorambucil, an oral medication, has single agent activity
`against chronic lymphocytic leukemia (CLL) and small B-cell
`lymphomas.'"
`Originally described in 1963, bendamustine (Fig. 14A-3) has
`emerged as an effective treatment for patients with CLL and indo-
`lent NHL.'''
`
`Aziritlines
`
`The stable aziridincs are analogs of the reactive ring-closed inter-
`mediates of the nitrogen mustards. Compounds hearing two or
`more aziridine groups, such as thiotepa (Fig. 14A-3; [thiotepa, tri-
`ethylenethiophosphoramidep, have clinical activity against breast
`and ovarian cancer,' but thiotepa is currently used as an occasional
`component of high-close regimens.' It was originally tested for
`antitumor activity because the nitrogen mustards alkylate through
`an aziridine intermediate. Both thiotepa and its primary desulfu-
`rated metabolite TEPA (triethylenephosphoramide) have cytotoxic
`activity in vitro.
`Altretamine, with hydroxyrnethylmelamine as the active metab-
`olite, is only rarely used as salvage therapy in recurrent ovar-
`ian cancer.' It is less toxic than other alkylating drugs but has a
`low level of antitumor activity for this disease. Although the
`mechanism of action of these compounds has not been explored
`thoroughly, they presumably alkylate through opening of the
`aziridine rings, as shown for the nitrogen mustards. The reactiv-
`ity of the aziridine groups is increased by protonation and thus is
`enhanced at the low pH more characteristic of tumors than normal
`
`tissues.
`
`Alkyl Maine Sultanates
`
`The major clinical representative of the alkyl alkane sulfonates is
`busulfan, which is widely used in high-dose regimens for the treat-
`ment of acute myelogenous leukemia.' Of the alkyl alkane sul-
`fonates, compounds with one to eight methylene units between
`the sulfonate groups have antitumor activity, but maximal cross-
`linking and activity are achieved by compounds with four units.'
`The mechanism of action of the alkyl alkane sulfonates is shown in
`
`Figure 14A-4.
`Busulfan exhibits second-order alkylation kinetics. The com-
`pound reacts more extensively with thiol groups of amino acids
`and proteins' than do the nitrogen mustards, and these findings
`have prompted the suggestion that the alkyl alkane sulfonates may
`exert their cytotoxic activities through such thiol reactions along
`with interactions with DNA.'" Brookes and Lawley35'36 were
`able to demonstrate the reaction of busulfan with the N-7 posi-
`tion of guanine. The cytotoxic potential of busulfan correlates with
`adenine-to-guanine cross-linking." Busulfan is markedly cyto-
`toxic to hematopoietic stem cells. This effect is seen clinically in
`the prolonged aplasia that may follow busulfan administration and
`can be shown experimentally in stem cell cloning systems." The
`pharmacologic basis for this property of busulfan is riot well under-
`stood but may involve damage to the mesenchymal stem cells in
`the microenvironment. In recent years, an intravenous formula-
`tion has simplified the dose appropriate administration of busul-
`fan to achieve optimal blood levels during high-close mycloablative
`treatments.
`
`11112 . = (cid:9)
`
`jai
`
`0
`O
`II
`II (cid:9)
`CH3-S -0 -CH2CH2CH2CH2 -0-S -CH3 + R-NH2
`II (cid:9)
`II
`0
`O
`
`R
`H-N-H
`O
`cH3-s-o-CH2CH2CH2CH2
`O
`o-s-cH3
`O
`
`0
`H (cid:9)
`O
`II
`II (cid:9)
`I (cid:9)
`CH3-5-0-CH2CH2CH2CH2 -N -R + H+ + -0-S-CH3
`II
`II (cid:9)
`O
`0
`
`FiGum: (cid:9)
`Structure and alkylating mechanism of busulfan, an alkane
`sulfonate. From Colvin M. Molecular pharmacology of alkylating agents. In: Cooke
`ST, Prestayko AW. Cancer and Chemotherapy, vol 3. New York: Academic Press,
`1981:291.)
`
`Ifitrosoureas
`
`The nitrosourca antitumor agents were discovered in a drug screen-
`ing effort that focused on analogues of methylnitrosoguanidine and
`methylnitrosourea.' Chloroethyl derivatives such as chloroethyIni-
`trosourea and BCNU (carmustine) (Fig. 14A-5) possess marked anti-
`tumor activity and had activity against tumor in the CNS."'" In
`addition to chloroethyl alkylating activity, the available nitrosoureas
`can also carbamoylate nucleophiles;" Closely related methylatincr
`agents, procarbazine, temozolomide, and clacarbazine (DTIC), are lipo -
`philic and penetrate the CNS (see nonclassical alkylating agents,
`this chapter).
`The nitrosoureas exhibit only partial cross-resistance with other
`alkylating agents,' and a number of studies established unique aspects
`of the mechanism of the alkylation reaction for these compounds
`(Fig. 14A-6). BCNU cross-links DNA after the formation of initial
`monoadducts, particularly at the N-7 position of guanine. As shown
`
`Chloroethylnitrosourea
`0
`CICH2CH2NCNH-R
`NO
`
`BCNU
`
`R = CH2CH2CI
`
`CCNU
`
`/CH2- CH2\
`CH2
`R = -CH (cid:9)
`\CH2-CH(
`
`/CH2- CH2\
`CH2-CH3
`Methyl CCNU R = -CH (cid:9)
`CH2-CH(
`
`FIGIL (cid:9)
`Structures of nitrosoureas. BCNU, bischloroethylnitroso-
`urea; CCNU, cyclohexylchloroethylnitrosourea.
`
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`

`
`Cellular Pharmacology (cid:9)
`
`271
`
`O
`II
`N H
`I I (cid:9)
`OH—
`CICH2CH2NCNCH2CH2CI ---"- CICH2CH2N=N—OH + O=C=NCH2CH2CI
`il
`O
`
`NH
`
`[C1CH2CH2+]
`
`Alkylation of nucleoside by bis-
`chloroethylnitrosourea (BCNU).
`
`in Figure 14A-6, the diazonium hydroxide intermediate formed
`during BCNLI hydrolysis decomposes to form a 2-chloroethyl
`carbonium ion (or equivalent), a strong electrophile, capable of
`alkylation of guanine, cytidine, and adenine bases.' In a subsequent
`step occurring over hours, the chloride is displaced by electron-
`rich nitrogen on the complementary DNA strand base to form a
`cross-link. DNA-protein cross-links are also possible by initiating
`chloroethylation at the amino or sulfhydryl group of protein.'
`Isocyanates resulting from the spontaneous breakdown of many
`of the methyl- and chloroethylnitrosoureas are also shown in
`Figure 14A-6. The role of isocyanate-mediated carbamoylation
`in antitumor effects is incompletely understood, but this activity
`may be responsible for some toxicities associated with nitrosourea
`therapy:"
`High-dose BCNU, etoposide, and cisplatin comprise the BEP
`regimen used for autologous stem-cell transplantation in patients
`with refractory or relapsed lymphoma:" Another high-dose
`BCNU-containing regimen, BEAM (BCNU, etoposide, cytara-
`bine, melphalan), has also been used with success with autologous
`hematopoietic stem-cell transplant in patients with NHL.' In the
`1980s, BCNU attracted interest as an adjuvant to radiation therapy
`in the treatment of patients with grade III and IV astrocytoma' but
`has been replaced by temozolomide." BCNU-impregnated polymer
`wafers implanted in the tumor bed at the time of surgical resection
`provide a controlled release form of local chemotherapy:"
`Streptozotocin is a unique methylnitrosourea with methylating
`activity that lacks carbamoylating activity. It is used exclusively in
`the treatment of metastatic islet cell carcinoma of the pancreas and
`malignant carcinoid tumors.' The dose-limiting toxicities in humans
`have been gastrointestinal and renal, but not hematopoietic.
`
`Alkylating Agent—Steroid Conjugates
`
`Steroid receptors may serve to localize and concentrate
`appended drug species in hormone-responsive cancers.
`A number of synthetic conjugates of mustards and steroids
`have been developed. Of these drugs, two have made the
`transition into clinical application. Prednimustine, an
`ester-linked conjugate and slow release form of chlorambucil
`and prednisolone, is no longer available for clinical use.'
`Estratnustine is a carbamatc ester—linked conjugate of
`nornitrogen mustard and estradiol but functions as an
`inhibitor of tubulin polymerization (see Chapter 13).
`
`Prodrulls of Alkylating Agents
`
`Therapy with alkylating agents is compromised by a high level
`of toxicity to normal tissues and a lack of tumor selectivity.
`Cyclophosphamide and ifosfamide were prodrugs synthesized
`in the hope that high levels of phosphamidases in epithelial
`tumors would selectively activate the drugs.' Strategies for
`more selective delivery of alkylating agent to tumor have
`been explored including cleavable tumor-directed antibody-
`alkylating agent conjugates," alkylating agent-glutathione
`conjugates (which might be selectively cleaved by glutathione
`transferase (GST) P1 expressed in high levels in tumor cells)"
`or viral vectors delivering activating enzymes to tumor cells.'
`
`Cellular Pharmacology
`
`Cellular Uptake
`
`The uptake of alkylating agents into cells is an important determi-
`nant of cellular specificity. Many are highly lipid soluble (including
`the active metabolites of the methylating agents, cyclophosph-
`amide, and ifosfamide, as well as chlorambucil) and readily enter
`cells by passive diffusion. Mechlorethamine uptake depends upon
`the choline transport system.' Melphalan is transported into sev-
`eral cell types by at least two active transport systems, which also
`carry leucine and other neutral amino acids across the cell mem-
`brane.''" High levels of leucine in the medium protect cells from
`the cytotoxic effects of melphalan by competing with melphalan
`for transport." In contrast to mechlorethamine and melphalan, the
`highly lipid-soluble nitrosoureas BCNU and CCNLI enter cells by
`passive diffusion.' Chlorambucil uptake also occurs through simple
`passive diffusion.
`Studies of cellular uptake of alkylating agents that require meta-
`bolic activation (such as cyclophosphamide or ifosfamide) arc ham-
`pered by uncertainty about which metabolite, or even parent drug,
`is the most critical moiety for transport.
`
`Sites of Alkylation
`
`Any alkylating agent producing reactive intermediates binds to a
`variety of cellular constituents' including nucleic acids, proteins,
`amino acids, and nucleotides. As an example, the active alkylating
`
`
`Ti:- (cid:9) Hats ial'
`L":
`
`ME'y
`
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`

`
`272 (cid:9)
`
`Chapter 14A • Classical Alkylating Agents
`
`species from a nitrogen mustard demonstrates selectivity for
`nucleophiles in the following order: (a) oxygens of phosphates,
`(b) oxygens of bases, (c) amino groups of purines, (d) amino
`groups of proteins, (e) sulfur atoms of methionine, and (f) thiol
`groups of cysteinyl residues of glutathione." This ranking, however,
`assumes there arc no steric or hydrophilic/hydrophobic barriers
`to the tissue nucleophile, and this is seldom the case. In addition,
`glutathione conjugation is often favored in the presence of GSTs,
`which offer catalysis. Thus, generalizations about alkylating agent
`targets are fraught with difficulty. In addition, it seems likely that
`a matrix of biochemical targets of alkylating agents may contribute
`to cytotoxicity, though DNA is generally favored as the primary
`target. Proof of this hypothesis may be emerging from three areas
`of research where cytotoxicity correlates with (a) activity of DNA
`repair enzymes, perhaps best shown for BCNU and repair by alkyl
`guanine alkyltransferase (AGT),63 (b) changes in a matrix of genetic
`and epigenetic events measured and analyzed by gene expression
`arrays,' and (c) specific DNA adducts shown by mass spectromet-
`ric analysis." The stringency of such analyses requires that alterna-
`tive toxic pathways not involving DNA must be excluded, a difficult
`requirement to meet. For this reason, mechanistic understanding of
`alkylating agent activity must be considered incomplete.
`In the DNA molecule, the phosphoryl oxygens of the sugar
`phosphate backbone are obvious electron-rich targets for alkyla-
`tion. Alkylation of the phosphate groups occurs"'" and can result in
`strand breakage from hydrolysis of the resulting phosphotriesters.
`Although the biologic significance of the strand breakage caused by
`phosphate alkylation remains uncertain, the process is so slow that
`it seems unlikely to be a major determinant of cytotoxicity, even for
`monofunctional agents.