Chapter 15
`
`Principles of Cancer Chemotherapy
`
`J.S. MALPAS and A. ROHATINER
`
`Section I: Historical Perspective
`
`Pharmacology of Anticancer Drugs
`Pharmacokinetic Principles
`Pharmacology of Alkylating Agents
`Mechanisms of Alkylation
`Pharmacology of Antimetabolic Drugs
`Methotrexate
`S-Fluorouracil
`
`Purine Antimetabolic Agents
`Pharmacology of Drugs Derived From Natural Sources
`Bleomycin
`Plant Alkaloids
`
`Taxol
`
`Epipodophyllotoxins
`Platinum Analogues
`The Use of Drugs for Cancer Treatment
`
`Objectives
`Diagnosis
`
`Advances in Oncobiology
`Volume 1, pages 317—350.
`Copyright © 1996 by JAI Press Inc.
`All rights of reproduction in any form reserved.
`ISBN: 0-7623-0146—5
`
`317
`
`318
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`320
`320
`323
`323
`326
`326
`327
`
`329
`331
`334
`335
`
`336
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`337
`3 37
`338
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`338
`339
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`ALVOGEN, Exh. 1059, p. 0001
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`318
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`LS. MALPAS and A. ROHATINER
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`Staging
`Drug Resistance
`Combination With Other Treatment Modalities
`
`New Principles
`
`Drug Toxicity
`Drug Resistance
`Mechanisms of Cell Resistance
`
`Induction of Drug Resistance
`Multidrug Resistance
`
`Topoisomerase
`Glutathione
`
`Section II: Effects of Drugs on Cells
`Colony Forming Assays
`In Viva/In Vitro ASSay
`In VIVO Assays
`
`Spleen Colony Assay
`Survival Curves
`
`Prediction of Response
`Use of Xenografts
`
`Summary
`
`340
`340
`341
`
`341
`
`341
`342
`343
`
`344
`344
`
`344
`345
`
`345
`345
`346
`347
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`347
`348
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`348
`348
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`349
`
`SECTION I: HISTORICAL PERSPECTIVE
`
`Although the chemotherapy of cancer is a relatively recent development, it must
`not be forgotten that even before the 19th century efforts were made, using various
`
`metals including zinc, silver, and mercury, in an attempt to treat cancer, and some
`
`success was reported in folk medicine from the use of either metals or plant extracts
`
`such as colchicine. It was not until 1865 that Lissauer reported the beneficial effect
`
`ofpotassium arsenite in chronic leukemia. Although the use of metals did not return
`
`for more than 100 years, when the platinum compounds were successfully intro-
`duced into cancer chemotherapy, a concept was born which was strengthened by
`the successful use of chemotherapeutic agents, first against protozoa and later
`against bacteria.
`The modern era of chemotherapy begins with the introduction of nitrogen
`
`mustard by Wilkinson in Great Britain and Gilman and Goodman in the United
`
`States. Significant clinical responses were obtained in patients with Hodgkin’s
`
`disease, when for the first time it was shown that a chemotherapeutic agent could
`
`affect a malignancy which had become widely disseminated. Until then, surgery or
`radiotherapy were the only available treatments to localize disease, and once cancer
`
`had spread, the patient inevitably died.
`
`It must be said that part passu with
`
`chemotherapy, the use of hormones was shown to be effective. Dodds synthesized
`
`stilbestrol and used it in disseminated prostatic cancer, and Hickman and Kendall
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`Principles of Cancer Chemotherapy
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`319
`
`introduced cortisone for lymphoid malignancies in 1949. New derivatives of
`
`nitrogen mustard were synthesized by Ross at the Chester Beattie Institute in
`London, and melphalan, chlorambucil, and myeloran were introduced to clinical
`
`practice by Haddow, Galton, and others.
`There still seemed no possibility of treating acute leukemia until the synthesis of
`
`the folic antagonist aminopterin by Seeger and the demonstration by Farber and his
`colleagues, in 1948, that it could produce remission in children with acute leukemia.
`These new substances, called antimetabolites, relied on an increasingly sophisti-
`
`cated knowledge of cell metabolism, and an ability to synthesize analogues of
`
`purines and pyrimidines. The work of Hitchings and Elion ushered in a golden age
`
`of chemotherapeutic development. Among the many antimetabolites produced,
`
`5—fluorouracil (synthesized by Heidelberger) stands out as a drug that was specifi-
`cally designed to treat carcinoma, and today remains one of the most effective
`
`agents available.
`Advances in the knowledge of biochemistry led to some interesting attempts to
`exploit the biochemistry of the tumor cell. It was thought that the essential amino
`acid phenylalanine might enable a drug attached to it to gain easier entry to the cell.
`This was the reason for the synthesis of melphalan, and although the principle did
`
`not work, nevertheless a very usefill chemotherapeutic agent was produced. The
`
`same was true ofthe compound cyclophosphamide, which is split by phosphatases
`present in high quantity in tumors. The high local phosphatase content was
`supposed to liberate the cyclophosphamide locally, and avoid damage to local
`
`tissues. Unfortunately this hypothesis was not borne out in practice, but neverthe-
`less cyclophosphamide has remained an important alkylating agent. A number of
`other drugs were introduced by serendipity: plant, bacterial, or fungal molds
`
`became a source of a wide variety of important compounds, many effective
`antibacterial agents. Drugs such as actinomycin D, daunorubicin, and doxorubicin,
`
`were developed by an increasingly sophisticated pharmaceutical industry which
`
`was aware of the potential of these compounds as anticancer agents. Serendipity
`also came to the aid of the chemist, when (for example) extracts of the Madagascar
`periwinkle were being examined as a possible antidiabetic agent, and were shown
`
`to reduce the white cell count in rabbits. Inhibition of growth of tumor cells was
`noted, and the two compounds vinblastine and vincristine were extracted. These
`
`remain two of the most potent and widely used anticancer agents.
`
`Along with the development of new agents came the realization of the best mode
`
`of their employment. Higher rates of response and more durable remissions could
`be obtained by using drugs in combination, particularly if the agents had different
`
`specific toxicities. Thus, while the antitumor effect sumrnated, the toxic side-effects
`
`(which were the main disadvantage of chemotherapy) were limited. The highly
`
`successful MOPP regimen introduced by DeVita and his colleagues for the treat-
`
`ment of Hodgkin’s disease, combinations of anthracyclines and alkylating agents
`for the treatment of childhood solid tumors, and the combination of platinum
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`320
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`LS. MALPAS and A. ROHATINER
`
`compounds with bleomycin and etoposide, resulting in the cure of testicular cancer,
`are some examples of the successful use of curative combinations of drugs.
`Although the history of chemotherapy is relatively short, major strides have been
`made towards the control of many previously fatal malignant conditions.
`
`PHARMACOLOGY OF ANTICANCER DRUGS
`
`Pharmacokinetic Principles
`
`Before considering the main classes of anticancer drugs. it is necessary to review
`the principles by which these drugs achieve their effects, and the factors which
`govern their absorption, distribution, and excretion: their pharrnacokinetics. Anti-
`
`cancer drugs, when administered orally, may be wholly or partially absorbed.
`Absorption may be influenced by a number of factors. The blood level of the drug
`then rapidly rises. During its passage through the liver it may undergo metabolic
`changes—so-called first pass metabolism—and various metabolites may start to
`circulate, being removed either locally or by excretion in the urine. in general, the
`
`blood level achieved will give an indication of the exposure of the tumor to the
`
`anticancer agent. The effective exposure will be a function of concentration
`
`ConcentrationofDrug
`
`Time
`
`: Area under the Curve (AUC)
`
`Figure 1. Curve showing variation of concentration ofdrug with time and derivation
`of the area under the curve (AUC).
`
`ALVOGEN, Exh. 1059, p. 0004
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`Principles of Cancer Chemotherapy
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`321
`
`ConcentrationofDrug
`
`Time
`
`Figure 2. Curve showing variation of concentration of drug with time when drug
`enters two separate compartments.
`
`multiplied by time. A typical exposure of a drug rapidly absorbed, uniformly
`distributed, and completely excreted, showing the curve for concentration and time,
`is given in Figure 1. The exposure of the tumor to the drug is measured by the area
`under the curve (AUC), and is shown in the shaded area in the figure. If the drug
`
`is distributed between two compartments, the curve of concentration in the blood
`will be modified (Figure 2). The rate of initial excretion is described as the time for
`half the drug to be excreted (tl/ZOL), and the time for the slower rate of excretion is
`tl/zfi.
`Bioavailability of a drug is a frequently used term. This can be measured by
`assessing the AUC for the intravenously administered dose ofthe drug, and dividing
`
`this into the AUC for the same oral dose of the drug.
`
`Bioavailabilit
`
`
`AUC for oral dose
`y _ AUC for i.v. dose of drug
`
`It can be shown that in the case of some drugs such as melphalan, an alkylating
`agent, there can be a wide variation in bioavailability, ranging from 10 to 50%, even
`within an individual. The same is true for 6-mercaptopurine and this may be of
`importance when long—term oral mercaptopurine therapy is being used for the
`treatment of childhood acute lymphoblastic leukemia.
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`322
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`LS. MALPAS and A. ROHATINER
`
`End of
`infusion
`
`l 0'3
`
`' ‘1',
`
`MTX
`
`level
`[M]
`
`5
`
`10
`
` 10'4
`
`10'6
`
`Severe
`
`toxicity
`
`Mild
`
`toxicity
`
`W 0
`
`50
`
`60
`
`70
`
`80
`
`10
`
`20
`
`30
`
`40
`
`10'5
`
`Figure 3. Curve showing poor excretion of methotrexate in a patient compared to
`the normal range.
`
`Time (hours)
`
`As well as giving an indication ofthe exposure ofthe tumor to the drug, the AUC
`
`is a measure of the exposure of normal tissues to the drug. Many chemotherapeutic
`
`agents are toxic to the bone marrow, and it can be shown that there is a close
`
`correlation between the increasing AUC and lowered platelet and white blood cell
`counts.
`
`For a drug that is mainly eliminated by renal excretion, deterioration in renal
`
`function may lead to an increased AUC. Methotrexate, for example, is largely
`excreted Via the kidneys, and a practical example of how delayed excretion could
`
`raise the AUC and give rise to possible life-threatening toxicity is shown in Figure
`
`3. Excretion and metabolic degradation of cytotoxic drugs may be influenced by
`
`many other factors, including the co—administration of other anticancer drugs,
`
`nonspecific medication used to control patients’ symptoms, etc. Even the sequence
`
`in which certain anticancer drugs are used may inhibit or enhance the action of a
`
`specific compound.
`
`ALVOGEN, Exh. 1059, p. 0006
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`ALVOGEN, Exh. 1059, p. 0006
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`Principles of Cancer Chemotherapy
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`323
`
`PHARMACOLOGY OF ALKYLATING AGENTS
`
`Alkylating agents were historically the first group of anticancer drugs to show
`consistent beneficial effect. From the beginning they have maintained their impor-
`tance in the armamentarium available, and have shown their versatility by their
`adoption in high-dose chemotherapy schedules (dealt with later).
`
`Mechanisms of Alkylation
`
`The structure of mechlorethamine (nitrogen mustard) is shown in Figure 4. The
`ethylinamine group CHZCHZCI is a highly reactive component which reacts with
`so—called nucleophilic elements in a wide variety of biological molecules. A
`reiatively simple example is shown in the reaction with the amino acid alanine
`(Figure 5).
`
`Monofunctional alkylating agents are those with a single CHZCHZCI grouping
`able to perform reactions such as those shown in Figure 5. Bifunctional alkylating
`agents are those with two ethylinamine groups, which, in addition to causing
`damage and breaks in DNA, can bridge and form cross—links in the double helix.
`
`It can be shown that one of the commonest bonds attached is that of guanine, and
`that it is specifically alkylated at the seventh nitrogen (Figure 6). The bond formed
`
`between two guanine molecules is shown in Figure 7.
`
`Because the parent substance mechlorethamine was a sclerosant, it was not
`possible to give it orally. Patients had to be admitted for therapy at great inconven-
`ience, and were often subject to severe and intractable vomiting. A search was
`therefore made for oral preparations which would be better tolerated. This led to
`the introduction of chlorambucil and melphalan (Figure 8). The addition of the
`modified ring structure to the nitrogen mustard moiety leads to greater stability.
`These drugs can be administered orally, although their bioavailability may vary
`considerably. Besides giving stability, it was thought that in some cases the addition
`of an essential amino acid such as phenylalanine would result in the agent being
`more avidly taken up by the tumor cells. In practice this was not the case, but
`nevertheless melphalan remains a very important alkylating agent. Another inter-
`esting idea was that the cleavage of cyclophosphamide at the site of the phosphorus
`atom by the high tumor content of phosphatase and phosphorylase might work in
`
`H3O _ N
`
`/ CH2 CH2 cw
`
`CH2 CH2 Cl
`
`Figure 4. Methlorethamine (nitrogen mustard).
`
`ALVOGEN, Exh. 1059, p. 0007
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`

`324
`
`LS. MALPAS and A. ROHATINER
`
`/ CH2 CH: C!
`
`C”? W”? 0‘
`
`CH3N
`
`CH CH Cl
`2
`2
`
`—> CH3N —- CH2
`I
`CH2
`
`CH2 CH2 Cl
`I
`
`CH2 CH2 Cl
`l
`
`/ I
`CH2
`NH2 CHCOOH
`(ALANINE)
`
`Figure 5. Alkyiation of an amino acid.
`
`CH3
`
`l
`
`Cl CH2 CH2 — N —_ CH2 CH2'
`\
`N7
`
`C/
`\ N
`H
`
`CH3
`I
`
`+ HCI
`
`0
`
`NH
`
`NH2
`
`N é
`
`Figure 6. Aikylation of guanine at the N7 position.
`
`
`
`Figure 7. Alkylation with a bond formed between the two guanine moiecules.
`
`ALVOGEN, Exh. 1059, p. 0008
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`Principles of Cancer Chemotherapy
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`325
`
`HOOC —— CH2 — CH2
`
`CHLORAMBUCIL
`
`HOOC —— CH2 — 01-12
`
`N
`
`N
`
`/ CH2 CH2 Cl
`
`\ CH2 CCH2 Cl
`
`/ CH2 CH2 cu
`
`\ CH2 CH2 Cl
`
`Figure 8. Structure of chlorambucil and melphaian which can be absorbed orally,
`compared with mustine hydro—chloride, which cannot.
`
`MELPHALAN
`
`practice to produce high local concentration of the antitumor agent (Figure 9). It is
`
`now known, of course, that cyclophosphamide is inert, and only becomes active
`
`when it
`
`is converted to 4-hydroxycyclophosphamide on first-pass metabolism
`
`(Figire 10).
`Although the original concept of the action of cyclophosphamide is faulty,
`
`nevertheless this drug, too, remains an extremely effective alkylating agent. It is
`
`important to note that not only do some of the metabolic products (such as
`
`phosphoramide mustard) have anticancer effects, but other metabolites such as
`
`acrolein produce toxic effects on the lining of the bladder, giving rise to hemor-
`
`rhagic cystitis (and possibly, ultimately, carcinoma) which is a feature of the use of
`
`this agent. 4-hydroxycyclophosphamide has been found to be active in vitro, and
`
`ALVOGEN, Exh. 1059, p. 0009
`
`CH2CH2CI
`
`/
`
`H N
`
`0 *
`\II
`P —— N
`
`\ CH2 CH2 0:
`
`* SITE OF ACTION OF PHOSPHATASE
`
`Figure 9. Site of action of phosphatase.
`
`ALVOGEN, Exh. 1059, p. 0009
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`

`326
`
`OH
`
`LS. MALPAS and A. ROHATINER
`
`N 0
`\ll
`P —-- N
`
`/
`
`CH2 CH2 Cl
`
`/
`0
`
`\ CH2 CH2 cn
`
`—>
`
`N
`\
`
`/
`o
`
`P —- N
`
`CH2 CH2 CI
`
`/
`
`\ CH2 CH2 Cl
`
`CYCLOPHOSPHAMIDE
`
`4 HYDROXYCYCLOPHOSPHAMEDE
`
`Figure 10. Formation of 4—hydrocyclophosphamide, the active derivative of cyclo—
`phosphamide.
`
`has made a useful contribution to the removal of malignant cells in bone marrow
`being prepared for autologous transfusion. It has been found to be between three
`and four times more toxic to tumor cells than to surrounding myeloid precursor
`cells, and consequently purging or cleaning of bone marrow is possible.
`Many other agents are known to have alkylating properties. Some of these, such
`as busulphan, have established a powerful role in the management of specific
`conditions such as chronic myeloid leukemia, while others such as the nitrosoureas
`demonstrate wide-ranging activity against a variety of solid tumors, including
`lymphomas, colon cancer, and brain tumors. After a quiescent period, alkylating
`agents are again becoming of interest because of the application of new techniques
`of structural analysis which allow the design of more effective agents and their
`development for clinical use. The production of drugs targeted on defined nucleo-
`tide sequences may open the way for more effective agents. New information about
`
`the repair mechanisms underlying the resistance that is acquired by tumor cells
`treated with alkylating agents may also be helpful. Specific methods of inhibiting
`these repair processes are now being developed.
`
`PHARMACOLOGY OF ANTIMETABOLIC DRUGS
`
`Antimetabolic drugs aim at disruption of essential metabolic pathways concerned
`in the manufacture of DNA or RNA. They work by competitive inhibition of key
`enzymes in these metabolic pathways. The most important drugs in this group
`include methotrexate, S-fluorouracil, 6-mercaptopurine, and cytosine arabinoside.
`
`Methotrexate
`
`Methotrexate was the most successful of a number of analogues. They were
`designed to inhibit the enzyme dihydrofolate reductase (DHFR). The essential role
`of vitamin 8,2 and folic acid in cell metabolism had been established earlier, and
`in looking for an antagonist to folic acid, methotrexate was produced. This was
`
`in 1948 to produce dramatic but transient remission in
`shown by Farber et al.
`childhood acute lymphoblastic leukemia. Extremely tight, irreversible binding of
`methotrexate to DHFR inhibits the pathway (Figure 1 l). The pool of reduced folate
`is required for the production of thymines and eventually DNA. Although metho-
`
`ALVOGEN, Exh. 1059, p. 0010
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`ALVOGEN, Exh. 1059, p. 0010
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`Principles of Cancer Chemotherapy
`
`327
`
`DIHYDROFOLATE
`REDUCTASE
`
`FH2
`
`l
`
`FH4
`
`Figure 11 . Block on the production of tetrahydrofolate production by methotrexate.
`
`trexate is exceedingly effective at blocking the activity of DHFR, it only requires
`a few molecules of the enzyme to return to activity to enable this metabolic block
`to be overcome. The administration of purines or a source of reduced folate such
`as 5-formyltetrahydr0folate (leucovorin or folinic acid) will overcome the metabo-
`lic block and rescue normal cells that have been exposed to methotrexate.
`Methotrexate is remarkable in that there is good bioavailability and it can be
`given safely intravenously, intramuscularly, and intrathecally. Care needs to be
`taken if there are large additional compartments in which the drug can be taken up
`and sequestrated, and then slowly released. Examples of this are the presence of
`ascites or large pleural effusions. The drug enters this third compartment and is
`released slowly. If rescue with leucovorin is stopped prematurely while this slow
`
`release is occurring, severe toxicity and even death may result.
`
`Methotrexate was the first drug to cure experimental leukemia in mice, to induce
`remission in childhood acute leukemia, to cure the rare solid tumor choriocarci—
`noma (when used alone), and to form part of the curative program for acute
`
`leukemia, used as continuation or maintenance therapy. It has a wide spectrum of
`activity and is relatively well tolerated. As has been noted above, it is excreted
`through the kidneys. Renal function therefore must be good if severe toxic side
`effects are to be avoided. Its short-term toxicity includes mucositis and bone
`marrow suppression, which is of fairly rapid onset and short duration. In more
`intense dosages, hepatotoxicity, pulmonary toxicity, and adverse effects on the
`
`central nervous system may occur.
`
`5-Fluor0uracil
`
`5-F1u0rouraci1 (SFU) is notable as an antimetabolite which was designed and
`synthesized to produce a particular biochemical effect to inhibit cell growth in
`tumor cells which were noted to have avidity for uracil. Up to the time of its
`production, most antimetabolites that had been produced were effective against
`leukemia or lymphoma. An antimetabolic agent with efficacy against solid tumors
`(particularly adenocarcinoma) was sought, and a team led by Heidelberger set out
`to produce such an agent. SFU remains a drug of great interest and versatility. It
`
`ALVOGEN, Exh. 1059, p. 0011
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`ALVOGEN, Exh. 1059, p. 0011
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`328
`
`‘ LS. MALPAS and A. ROHATINER
`
`o
`
`o
`
`N
`
`\ /uj
`0%3
`
`N
`
`\ fil:
`aka
`
`URACIL
`
`5 FLUOROURACIL
`
`Figure 12. The similar structures of uracil and 5-fluorouracil (5FU).
`
`achieves antitumor activity against breast cancer and colon adenocarcinoma. New
`knowledge ofbiochemistry has allowed its antitumor effect to be enhanced by using
`other agents in conjunction with it, as well as with radiotherapy.
`The structures of uracil and S-FU are shown in Figure 12 and the mechanism of
`
`action of 5FU is thought to work in two main ways. It is first converted to the
`
`nucleoside 5-fiuorouridine diphosphate and triphosphate (FUDP) (Figure 13).
`
`These are incorporated into RNA by the action of RNA polymerase. These specious
`compounds then interfere with RNA function. The other pathway is the conversion
`to fluorodeoxyuridine monophosphate (FdUMP) which competitively inhibits the
`
`enzyme thymidylate synthetase, thus preventing the production of thymidylate,
`
`essential for DNA synthesis.
`
`It has subsequently been found that sequential use of methotrexate enhances the
`toxicity of 5FU, and as might be expected, the addition of 5-formyltetra-hydrofolate
`(leucovorin) which enhances the reduced folate available, improves the antitumor
`activity of 5FU.
`
`5FU is catabolized to dihydrofluorouracil in the liver. Some 90% is eliminated
`
`by metabolism, and some 5% is excreted unchanged in the urine. It is also poorly
`
`absorbed, and is best given intravenously. Because of detoxification at first pass in
`the liver, continuous infusion is more effective and less likely to lead to toxic effects
`than bolus administration. However, prolonged infusion gives rise to the major
`toxic effect of myelosuppression and mucous membrane ulceration. Skin rashes,
`
`conjunctivitis, ataxia due to cerebellar damage, cardiotoxicity and peeling of the
`
`5FU --—) 5FUMP ——) 5FUDP — 5FUTP
`
`5FUTP —-) RNA
`
`Figure 13. Pathway of conversion of 5FU into compounds affecting RNA.
`
`ALVOGEN, Exh. 1059, p. 0012
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`

`Principles of Cancer Chemotherapy
`
`329
`
`soles of the feet and palms of the hands (the so—called hand-foot syndrome") have
`been reported, and these toxic side effects may be dose-limiting.
`
`Purine Antimetabolic Agents
`
`6-Mercaptopurine
`
`6-Mercaptopurine (6MP) and 6-Thioguanine (6TG) were among the earliest of
`the antimetabolic agents introduced at the beginning ofthe 19503. Their importance
`lies in the contribution they have made to the cure ofchildhood acute lymphoblastic
`leukemia, where their use combined with methotrexate as continuation therapy has
`been responsible for the high cure rate. Both 6MP and 6TG were found to induce
`remissions in acute lymphoblastic and myeloblastic leukemia. They were not
`effective in chronic leukemias or lymphoid malignancies. The exact manner in
`which 6MP achieves its effect is still unknown, although it must inhibit purine
`synthesis and interconversion. The same uncertainty exists for 6TG. The metabolic
`transformation of 6MP, though complex,
`is of note because of the effect that
`commonly prescribed xanthine oxidase inhibitors such as allopurinol may have on
`decreasing 6MP degradation. Doses of 6MP should be reduced by 50% when
`allopurinol is being given concurrently (Figure 14).
`Pharmacokinetics show a considerable variation in bioavailability. There is great
`interpatient variation in absorption, and controversy over whether the drug is best
`absorbed in the morning before food, or at night. The half-time range for elimination
`of drug given intravenously varies from 20 minutes to an hour, and the drug is
`almost entirely eliminated by metabolism, very little being excreted. If the drug is
`given by mouth. there is considerable first pass metabolism in the liver, but the
`half—life seems to be about three hours.
`
`6MP is now used mainly as continuation therapy in acute lymphoblastic leuke-
`mia. 6TG is preferred in induction regimens for acute non-lymphoblastic leukemia,
`
`6MP —> 6MP ribose phosphate —-> nucleic acid formation
`xanthine
`oxidase
`
`6 THIOXANTHINE
`
`xanthine
`oxidase
`
`6 THlOURIC ACID
`
`* Sites of block to 6MP oxidation and elimination which are
`enhanced by the xanthine oxidase inhibitor allopurinol
`
`Figure 14. Pathway of 6-mercaptopurine (6MP), showing the reason for reducing
`6MP dose when allopurinol is given.
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`330
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`LS. MALPAS and A. ROHATINER
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`and both have been used in the blast crisis of chronic myeloid leukemia. Myelo—
`suppression is the main toxic side effect, but in some patients a mild cholecystatic
`jaundice has been reported. However, this is usually reversible on withdrawal of
`the 6MP or 6TG. Nevertheless, care should be taken when either of these drugs is
`being administered in conjunction with other hepatotoxic agents, such as doxoru-
`bicin.
`
`Cytosine A rabinoside
`
`Another example of an antimetabolite which is effective in the treatment of
`leukemia is cytosine arabinoside, ara—C. This compound was of great interest, and
`following its introduction it was found to have efficacy against a highly resistant
`form of leukemia—acute myelogenous leukemia—which had otherwise responded
`poorly to antimetabolites such as 6MP and methotrexate. With ara-C the response
`
`rates rose to more than 50%, and when it was combined with anthracyclines (see
`
`below) further improvement in remission induction occurred, and these two agents
`
`have become the mainstay of treatment of acute myelogenous leukemia.
`
`Ara-C was originally isolated from a sponge, but is now produced synthetically.
`
`It is very similar in structure to deoxycytidine (Figure 15). The main difference is
`a hydroxyl group on the sugar, converting the latter from a deoxyribose to an
`arabinoside. The main event when ara~C enters the cytosol is its conversion to
`
`ara-CTP. This binds to DNA polymerase, stops DNA production, and hence arrests
`growth. Ara-C may also be incorporated into DNA, disturbing its synthesis by
`terminating DNA chain elongation. As can be seen from Figure 16, there are two
`
`N
`
`O
`
`O
`
`O
`
`N
`
`O
`
`HO
`
`HO
`
`HO
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`DEOXYCYTIDINE
`
`CYTOSINE ARABINOSIDE
`
`Figure 15. The similar structures of deoxycytidine and cytosine arabinoside.
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`Principles of Cancer Chemotherapy
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`331
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`ARAU(-- ARAC -—-> ARA CMP —-> ARA CTP
`
`hm
`CYTIDINE
`DEOXYCYTIDINE
`DEAMINASE
`KINASE
`
`Figure 16. Pathways for the breakdown of cytosine arabinoside by the enzymes
`cytidine deaminase and deoxycytidine kinase.
`
`mechanisms, both enzymic, which affect the amount ofara*CTP present, and hence
`the effectiveness of the drug. The kinases which produce ara-CTP, and the deami-
`nases which destroy it, are vital to its activity.
`
`Ara—C cannot be given by mouth, but is otherwise very versatile. It can be
`administered intravenously, intrathecally, and subcutaneously. This last route is
`becoming the route of choice for out-patient therapy, and since the half-life is so
`short, and the drug acts best on cells in the synthetic phase of the cell cycle, there
`
`are advantages in giving the drug by long-term subcutaneous infusion.
`Studies on high-dose ara-C have shown that parenteral dosage of cytosine allows
`cytotoxic levels of the drug to be achieved in the cerebrospinal fluid. This has been
`helpful in prophylaxis and treatment of meningeal leukemia. Very high doses of
`ara-C have produced (in addition to the usual myelosuppression and gastrointestinal
`toxicity) specific effects on the central nervous system, including damage to the
`cerebellum with nystagmus, severe ataxia, and eventually (if the drug is not
`stopped) central nervous effects on the cortex with dementia and coma. Early
`detection of nystagmus is helpful in avoiding these serious toxicities. Another
`unusual effect is the acute conjunctivitis produced by ara-C. This can be prevented
`
`by giving steroid eye-drops.
`After intensive investigation, the rationale for high—dose ara—C has been shown
`to be flawed, since the production of the effective ara—CTP is limited by enzymic
`process involving the kinases. Since these form a rate-limiting step in the metabolic
`pathway, increasing the dose of ara-C will not be followed by a greater antitumor
`effect.
`
`PHARMACOLOGY OF DRUGS DERIVED FROM NATURAL
`SOURCES
`
`A number of very potent cytotoxic drugs have been derived from natural sources
`such as streptomyces (doxorubicin, daunorubicin, actinomycin D, and bleomycin),
`plant products such as the vinca alkaloids from the Madagascar periwinkle, and
`epipodophyllotoxins from the mandrake plant. Many were found as the result of
`antitumor effects being noted when they were screened in the search for new
`antibiotics (for example). Among the most important are the anthracyclines. These
`have the basic formula shown in Figure 17. Relatively small structural changes
`distinguish doxorubicin, daunorubicin, and epirubicin, but their clinical activity and
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`332
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`LS. MALPAS and A. ROHATINER
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`— Fl
`
`HC
`
`0H
`
`O
`
`OH
`
`OFH2
`
`O
`
`OH
`
`HO
`
`
`
`9
`
`EPIRUBICIN
`
`HO
`
`0
`
`0
`
`CH
`
`
`
`H = CH2 OH DOXORUBICIN
`Fl = CH2
`DAUNORUBICIN
`
`Figure 17. Structure of doxorubicin and daunorubicin.
`
`to some extent their toxicities are widely different. Thus, while doxorubicin is
`effective in acute lymphoblastic and myeloblastic leukemia, it also has activity in
`solid tumors such as breast, ovary and lung cancer. It is used in lymphomas and in
`
`childhood malignancy. Daunorubicin, on the other hand, is a most effective agent
`
`against myeloblastic leukemia, and has little effect against other solid tumors. Both
`have serious cumulative cardiotoxicity, but this is less apparent in the analogue,
`
`epirubicin.
`
`Although in clinical use for many years, there is uncertainty as to their mecha-
`
`nisms of action. The anthracyclines can be shown to intercalate with DNA. This
`mechanism cannot account for various features oftheir antitumor activity, and other
`actions have been proposed. Recently a mechanism whereby anthracyclines bind
`to an enzyme called topoisomerase II, which promotes strand breakage and re-
`sealing, has been described, and is thought to be increasingly important. Adiagram
`of the action is shown in Figure 18.
`
`Anthracyclines can cause topoisomerase II-mediated DNA changes, and these
`effects are seen at concentrations of anthracycline which are achievable in patients.
`Supporting evidence for this theory comes from the fact that when topoisomerase
`II enzyme levels fall in patient tumors, anthracycline activity is inhibited, and the
`tumor becomes resistant to their effect.
`
`Another mode of action relates to the production of superoxide. Bacterial cell
`killing has long been known to be due to superoxide. Semiquinones, which form
`part of the anthracycline structure, are potent producers of superoxide. Superoxide
`can form the basis for the production of free radicals such as hydroxy groups, which
`are perhaps the most powerful cytotoxic moieties known. Unfortunately, in addition
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`Principles of Cancer Chemotherapy
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`333
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`DNA
`
`DNA
`
`DNA
`
`TOPO ll
`
`DOXORUBICiN
`
`TOPO II
`
`DOXORUBICIN
`
`Figure 18. Diagram of the mechanism of action of topoisomerase II.
`
`to their effect on the tumor cell, these free radicals, in conjunction with iron, have
`the ability to cause damage to the myoblast in the myocardium, and are thought to
`be related to the serious cumulative cardiotoxicity of both doxorubicin and daun—
`orubicin.
`
`Doxorubicin is one of the most potent sclerosing drugs known. Repeated long—
`
`term dosage, or administration as infusion, requires the use of a central venous line.
`This results in the drug being diluted, and reduces the incidence of extravasation
`and thrombosis. Doxorubicin has a short tl/zo. of about 10 minutes. Tl/zfi is three
`hours, but the tl/zy is very long, at more than 30 hours. ' This has a clinical
`implication, because the use of freezing caps to prevent the profound alopecia
`produced by the drug is not feasible when the drug circulates for such a long time.
`Bolus injection of doxorubicin results in high serum levels, and it appears that
`cardiotoxicity is related to these. Cardiotoxicity is reduced when the drug is given
`
`by infusion (Figure 19).
`Many attempts have been made to reduce the cardiotoxicity of anthracyelines by
`giving other agents. Among the most useful so far developed is ICRF187, which
`has been shown to reduce the number of cardiac events in women on long-term
`
`anthracycline treatment for breast cancer (Figure 20). Other toxic side-effects are
`
`myelosuppression, mucositis, hair loss, and severe local injury on extravasation (as
`mentioned above).
`Doxorubicin (adriamycin) is effective in a wide range of hematological and solid
`
`tumors. It has been a component in a number of well—establishe