Antineoplastic drugs: clinical pharmacology and therapeutic use.

Review Article

Drugs 16: 46-87 (1978) © AD IS Press 1978

Antineoplastic Drugs: Clinical Pharmacology and Therapeutic Use Richard A . Bender, Leonard A. Zwelling, James H. Doroshow, Gershon Y. Locker, Kenneth R. Hande, Donald S. Murinson, Michael Cohen, Charles E. Myers and Bruce A. Chabner Medicine Branch and Clinical Pharmacology Branch, National Cancer Institute, Bethesda, Maryland

The treatment of malignant disease has undergone an evolution over the past decade with the development of many new agents and combinations of agents useful in antineoplastic therapy. Such agents are somewhat unique from other classes of drugs in having a very narrow therapeutic index. Furthermore, as many of these agents only exert their effects during certain phases of the cell cycle, their action is schedule-dependent. Thus, a thorough understanding of the pharmacology of these agents and of potential drug interactions is imperative for their safe and effective use by the oncologist or cancer therapist. This review will deal with the clinical pharmacology of some of the more commonly used antineoplastic agents with emphasis on selected newer agents of proven therapeutic efficacy.

1. A lkylating Agents I. 1 Cyclophosphamide

The development of cyclophosphamide as a chemotherapeutic agent represents the culmination of efforts to design an alkylating agent with specificity for malignant cells. The structure of this compound is shown in figure I.

1.1.1 Metabolic Activation

Arnold and Bourseaux (1958) reasoned that tumour cells, which contained high levels of phosphamidase, would cleave the phosphoramide ring of this compound, producing a highly reactive phospho-mustard. In fact, it now appears that this ring cleavage takes place as one of aseries of activation reactions in the liver prior to entry of the active metabolite, aldophosphamide, into peripheral tissues. The current understanding of cyclophosphamide metabolism is shown in figure I, and includes: (I) hydroxylation of the phosphoramide ring by liver microsomes, (2) spontaneous ring opening to yield aldophosphamide, (3) non-enzymatic cleavage of aldophosphamide within peripheral target cells to yield the highly reactive species, phosphoramide mustard, and (4) urinary excretion of at least two minimally active metabolites, carboxyphosphamide and 4-ketocyclophosphamide, as well as lesser amounts of parent drug. In addition to phosphamide mustard, a second active principle, acrolein, is produced by aldophosphamide cleavage, "but acrolein is less potent in vitro as an antineoplastic agent and has unknown importance as an end product of cyclophosphamide metabolism (Fenselau, 1976).

Antineoplastic Drugs: Clinical Pharmacology and Therapeutic Use

liver ,microsomes

0 / O-CH 2

I'" CI-CH~H2

,II N-P

/ "NH-CH/ CI-CHrCH

CI-CH'2CH2

O/O-CH 2

"II

-,

N-P

CH2

'"NH-CHI /

CI-CH-CH /

2

2

47

2

2

CH2

OH cyclophosphamide

4-0H cyclophosphamide

t

spontaneous

CI~H2CH"

/

CI-CHrCH2

o

1I/0-CH2,

N-P

-,

aldophosphamide

~eriPheral ~~_~

./

tissues

r - - - - - - - - - - - - - - - - - - - , , ; spontaneous I I

CI-CH-CH 2

I

I

: I

I

0

0-

H-C

'11/

/"

CI-CH 2-CH2

~

2

N-P,

CH

+ NH 2

phosphoramide mustard

:

/

HC

II

0

acrolein

L

I I

degradation

NHr-C

/

CH2

~

~ivernon-microsomet . carboxyphosphamide 4-Keto phosphamide other alkylating metabolites

I

I : I I

: ~

Active Principles

Fig. 1. The metabolism of cyclophosphamide.

While this complex scheme of biotransformations has been elucidated in recent years, there exists at present only limited understanding of the relationship between the concentration or distribution of the various end products and the clinical toxicity of the drug. A second question of great importance is the possible influence of other pharmacological agents on the initial activation of cyclophosphamide by hepatic microsomes.

1.1.2 Drug Interactions Hepatic microsomal drug metabolising enzymes may vary greatly in activity depending on animal species and the effect of these enzymes on a particular drug interaction may also vary in different animal

tumour models, leading to conflicting information. Such conflicting data exists for the phenobarbitonecyclophosphamide interaction. Field and co-workers (1972) studied the effects of pretreatment with phenobarbitone or 2-diethylaminoethyl-2,2-diphenylvalerate (SKF 525A), an inhibitor of liver microsomal drug metabolising enzymes, on the survival and levels of alkylating metabolites in L1210 tumour-bearing mice treated with cyclophosphamide. Pretreatment with phenobarbitone accelerated production of the alkylating metabolites, followed by rapid depletion of the parent compound and its metabolites, and resulted in a shorter duration of the antileukaemic effect as measured by shortened survival times. SKF 525A pretreatment retarded activa-


tion of cyclophosphamide, increased the amount of alkylating metabolites present over a longer time duration, and lengthened the antileukaemic effect of cyclophosphamide. Sladek (1972) noted that pretreatment with phenobarbitone increased the rate of metabolism of cyclophosphamide and that pretreatment with 3-methylcholanthrene, thioacetamide, morphine or cobalt chloride depressed cyclophosphamide metabolism in rats bearing Walker 256 carcinosarcoma. Alberts and van Daalen Wetters (1976a,b) have recently shown that phenobarbitone pretreatment markedly reduced the antileukaemic activity of cyclophosphamide by reducing the total plasma alkylating metabolites, while allopurinol enhanced the effect of cyclophosphamide using the spleen colony assay system and survival studies in mice bearing P388 leukaemia. In summary, data for the phenobarbitonecyclophosphamide interaction in animal systems is confusing. Phenobarbitone probably reduces total cyclophosphamide alkylating metabolites, although 'peak' plasma levels for the active metabolites may actually be increased. Despite numerous animal studies which suggest that liver microsomal inducing agents alter cyclophosphamide activity, similar data in man are lacking. Bagley et al. (1973) reviewed the clinical pharmacology of radiolabelled cyclophosphamide in 26 patients. Although individual variation in plasma cyclophosphamide half-life and peak alkylating levels were noted following prior exposure to pentobarbitone, phenytoin, allopurinol or prednisone, the authors noted no significant difference in the total (concentration x time) product for a given cyclophosphamide dose. In contrast to most animal studies, they suggest that alterations in the rate of cyclophosphamide metabolism by other agents does not alter the toxicity or therapeutic effect. The effect of prednisolone on the activation of cyclophosphamide was studied by Hayakawa and co-workers (1969). Simultaneous administration of prednisolone inhibited activation of cyclophosphamide and decreased the ratio of active/ inactive metabolites in serum samples. However, Hanasono and Fischer

48

(1972) found no effect of corticosteroids on the plasma alkylating activity of cyclophosphamide. When chloramphenicol, an antibiotic which inhibits microsomal enzymes, was used to pretreat rats prior to cyclophosphamide therapy, the in vivo conversion of cyclophosphamide to the active alkylating metabolites was decreased. Chloramphenicol also increased both the mean day of death and the mean lethal dose of cyclophosphamide (Dixon, 1968). The effects of a number of compounds on cyclophosphamide activity and metabolism were studied in L1210 leukaemic mice and in human epidermoid carcinoma No.2 cells (Hill et al., 1972). Nicotine, atropine, ephedrine, apomorphine, and cocaine were potent inhibitors of the microsomal enzyme conversion of cyclophosphamide to its active metabolite, aldophosphamide, while cytochrome C, SKF 525A, and some steroid hormones were less potent inhibitors. Triiodothyronine or chlorpromazine combined with cyclophosphamide inhibited tumour growth more than cyclophosphamide alone (Bacigalupo, 1964). These studies demonstrate the effect of drugs often used clinically with cyclophosphamide on the metabolism of this agent. The relevance of these experimental studies to use of cyclophosphamide in man is uncertain, but an awareness of potential interactions of cyclophosphamide with other drugs, especially those with agents capable of inducing liver microsomal drug metabolising enzyme activity, may facilitate the clinician's perception of altered drug activity. Definitive studies in man await the development of a suitable specific assay for cyclophosphamide and its active metabolite, aldophosphamide, in plasma.

1.1.3 Pharmacokinetic Properties The human pharmacokinetics of cyclophosphamide have been elucidated in studies with radioactive-labelled cyclophosphamide. These studies reveal that unchanged parent drug and its metabolites, some with alkylating activity, appear both in plasma and urine of humans following drug administration by the oral or parenteral route (Mellett, 1971; Bagleyet


aI., 1973). The plasma disappearance curve in man, as well as in other species, is biphasic with an initial rapid distribution phase and a second slower phase representing metabolism and excretion. At doses ranging from 10 to 80mg/kg in man, the plasma half-life for cyclophosphamide has been reported to be in the range of 4 to 6.5 hours (Mellett, 197 1; Bagley et aI., 1973). The parent drug does not appear to be bound to plasma proteins, although 50 % binding has been reported for cyclophosphamide metabolites possessing alkylating activity. The renal clearance of cyclophosphamide is low, about II ml/min (15 % of creatinine clearance) and is increased to 18ml/min in phenobarbitone-pretreated patients (lao et aI., 1972). Since cyclophosphamide is not bound to plasma proteins, these low values indicate that extensive renal tubular reabsorption takes place. However, in patients pretreated with phenobarbitone or in non-pretreated patients, the total amount of parent drug (--- 10 %) and metabolites r.; 50 %) excreted in the urine during the first 24 hours is comparable and confirms the finding that only rates of excretion are affected by phenobarbitone pretreatment. Excretion of cyclophosphamide and metabolites in faeces and expired carbon dioxide amounts to less than 2 % in each case, and only minute amounts of the drug have been detected in the cerebrospinal fluid (CSF), breast milk, sweat, saliva and synovial fluid (Duncan et aI., I 973). 1.1.4 Adverse Reactions As might be expected from its similarity to other alkylating agents, cyclophosphamide exerts its primary toxic effects on rapidly dividing tissues, specifically on bone marrow cells and gastrointestinal epithelium, and has maximum cytotoxic activity for cells exposed during active DNA synthesis. Myelosuppression is primarily manifest as granulocytopenia 7 to 14 days after drug administration, with relative sparing of platelets. Alopecia is also a common side-effect. Sterile haemorrhagic cystitis, which is believed to be secondary to renal excretion of alkylating metabolites, occurs following high-dose infusions, or

49

more commonly with prolonged, low-dose administration as is used in the lymphomas, ovarian cancer, or multiple myeloma. Cystitis appears to result from chronic inflammation leading to fibrosis and telangiectasia of the bladder epithelium, and haemorrhage may become life-threatening if drug administration is continued. Intravesicular instillation of acetylcystine, a free radical scavenger, has been shown to reduce the incidence of cystitis in dogs, but as yet has not been used in man (Primack, 1971). In an effort to minimise the risk of haemorrhagic cystitis, forced diuresis is often attempted during high dose (> 50mg per kg) infusions of cyclophosphamide. However, with parenteral doses of 50mg/kg or greater, severe impairment of water excretion has been reported, leading to hyponatraemia, weight gain, and inappropriately concentrated urine, a toxic manifestation resembling the syndrome of inappropriate antidiuretic hormone secretion (DeFronzo et aI., 1973). Thus, in hydrated patients, it is important to be aware of the susceptibility to water intoxication and symptoms related to hyponatraemia. Serious cardiac toxicity has also been reported in patients with the use of high-dose cyclophosphamide therapy (Buckner et aI., 1972). Cyclophosphamide has been shown to cause sterility, fetal malformations and malignancies in experimental animals. Dysmorphogenicity has also been ascribed to cyclophosphamide in man, on the basis of isolated case reports (Sieber and Adamson, I 975). Carcinogenesis has also been suspected in man on the basis of individual cases; in addition a recent survey of alkylating agent-treated patients with ovarian cancer revealed an estimated 66.7 to 171.4fold increase in the incidence of acute myelogenous leukaemia (Reimer et aI., 1977). These late toxic effects are of particular significance in view of the growing use of cyclophosphamide in non-malignant diseases (severe rheumatoid arthritis, nephrotic syndrome, etc) and in adjuvant therapy of stage II breast carcinoma. Finally, the long-term complications of continuous immunosuppression by cyclophosphamide are of potential concern as well in the adjuvant setting (Mellett, 1971).


1.2 Cis-Platinum Cis (II) platinum diamminedichloride (cis-platinum) is the only inorganic compound in wide use in clinical oncology. In 1965, Rosenberg and his colleagues observed filamentous elongation of bacteria without cellular division when an electric current was applied to the medium in which the bacteria were growing (Rosenberg et al., 1965). Platinum from the system's electrodes had combined with chemical species in the medium to produce substances that affected these changes in the organisms. By 1969, several of these compounds, one of which was cisplatinum, had been isolated and found to be tumouricidal (Rosenberg et al., 1969). Since then, cisplatinum has demonstrated activity against testicular, ovarian, genitourinary, head and neck, paediatric, prostatic and osteogenic malignancies (Rozencweig et al., 1977b). 1.2.1 Mode of Action The structure of cis-platinum is shown in figure 2. Several important structural features are necessary for antitumour activity (Rosenberg, 1975; Thomson et al., 1972). Of key importance, however, is that the leaving groups be in the cis- as opposed to the trans-position. Additionally, the groups trans- to the leaving group, i.e. the -NH 3 moieties, should be bound tightly. The active form of cis-platinum is not the structure as depicted in figure 2, but rather an aquated form in which the 2 chloride leaving groups are replaced by 2 positively charged water molecules (Thomson, 1974). In vivo, the cis-platinum will circulate in its native state exchanging its own chloride

,..-

......., CI

Pt(ll)

NH3

a.. --- - - - - - - - --J

Fig. 2. The structure of cis-diamminedichloride platinum (II).

50

ligands, with the prevalent chloride ions in the blood thus remaining neutral. Upon entering a cell, the drug encounters a milieu of far lower chloride ion concentration. The chloride ligands then exchange for protonated water ligands and the molecule can react with intracellular constituents. Although the exact intracellular reaction that makes cis-platinum tumouricidal is unknown, the target of the drug is DNA. Cis-platinum inhibits cellular division in bacteria (Rosenberg et al., 1965), induces lysogenic phage from bacteria (Reslova, 1971), is mutagenic (Beck and Brubaker, 1975), and inactivates transforming DNA in bacterial systems (Munchausen, 1974). It has been shown to selectively bind to intracellular DNA and selectively inhibit DNA synthesis in several cell culture systems, including several human cell lines (Howle and Gale, 1970; Heinen and Bassleer, 1976; Harder and Rosenberg, 1970; Howle et al., 1971). However, cis-platinum displayed no cell cycle specificity in a synchronised line of human lymphoma cells (Drewinko et al., 1973). The active form of cis-platinum is a potential alkylating agent as several nucleophilic sites on the DNA bases would be susceptible to attack by the aquated drug. DNA interstrand cross-linking by cis-platinum has been demonstrated with spectrophotometric assays (Horacek and Drobnik, 1971; Harder, 1975) as well as alkaline cesium chloride sedimentation of density hybrid DNA (Roberts and Pascoe, 1972; Pascoe and Roberts, 1974). However, some authors feel that interstrand cross-linking is not the significant lethal lesion as they found it to be a relatively rare event which does not correlate with the biological activity of the drug in their studies (Munchausen, 1974; Shooter et al., 1972). However, no study of cisplatinum cross-linking to date has examined, in detail, the role of repair processes in antitumour activity (Beck and Brubaker, 1973; Van den Berg and Roberts, 1975, 1976).

1.2.2 PharmacokineticProperties The inorganic nature of cis-platinum has provided a singular difficulty in the study of its disposition and


cis-platinum 193m and cis-platinum 19 5m have been used (Lange et .al., 1972, 1973; Smith and Taylor, 1974; DeConti et al., 1973). A 2 phase disappearance curve from human plasma was demonstrated with rapid initial clearance (u /2 == 25.5 to 49.0 minutes) and a more prolonged second phase (u /2 == 58.5 to 73.0 hours). Although initial excretion was rapid it was not complete, as under 30 % of an administered dose was excreted in the first 24 hours and over 50 % was retained after 5 days. No preferential accumulation by tumour tissue was noted. 65 to 97 % of plasma radioactivity was associated with protein. The organs with the highest radioactivity were liver and kidney in animals and man. Atomic absorption spectrometry has allowed more prolonged studies as the radioactive forms of cis-platinum are shortlived. A study of beagle dogs revealed a 2-part disappearance curve similar to that described above with the additional observation that bile showed little platinum activity (Litterst et al., 1976). Liver and kidney were again noted as primary sites of accumulation, although ovary and uterus were also noted to have high concentrations of drug. Similar biphasic plasma disappearance curves were seen when cis-platinum was administered to humans with mannitol and frusemide (furosemide) diuresis (Higby et al., 1977). Bolus administration showed peak plasma levels at 30 minutes declining by first order kinetics, with a u /2 of 90 minutes. Urinary drug excretion peaked at 40 minutes with a t 1/2 of 60 minutes. 6· hour infusions resulted in peak plasma levels that were 15 % of that seen with an equivalent bolus dose. Cerebrospinal fluid levels were measured in 1 patient and found to be 2.5 % of serum levels after the first dose and 5.0 % after the second dose, showing some evidence for accumulation.

1.2.3 Adverse Reactions

Toxicity has been similar in all animal species tested, including man. The dose-limiting toxicity in man is nephrotoxicity. Early animal studies revealed renal proximal tubular damage similar to that seen

51

with mercurial compounds (Kociba and Sleight, 1971; Leonard et al., 1971), cystic lesions of the cortico-medullary junction (Ward and Fauvie, 1976) and glomerular lesions in monkeys (Stadnicki et al., 1975). Both acute and chronic nephrotoxicity have been described in humans being treated with cisplatinum (Higby et al., 1973; Talley et al., 1973; DeConti et al., 1973; Hardaker et al., 1974; GonzalezVitale et al., 1977), with significant reductions in creatinine clearance observed in patients receiving cisplatinum over a 6 to 12 month period (Dentino et al., 1977). Dose recommendations which derived from these studies were 1 to 2mg/kg or 50 to 100mg/m 2 • Recent work has provided evidence that prehydration as well as the' concomitant administration of frusemide and/ or mannitol with cis-platinum may ameliorate the nephrotoxicity of the drug (Ward et al., 1977; Hayes et al., 1977; Chary et al., 1977) although haematologic toxicity (vide infra) is not altered. This could allow higher doses to be administered.. In light of these newly developed methods of decreasing the risk of nephrotoxicity, adverse haematological effects are of increasing concern. Thrombocytopenia, panleukopenia, and reticulocytopenia were noted in animal studies (Schaeppi et aI., 1973; Zak et al., 1972a; Thompson and Gale, 1971). Dose-related myelosuppression has similarly been found in clinical trials (DeConti et al., .1 973; Talleyet al., 1973; Higby et al., 1973). Nausea and vomiting resistant to antiemetics and high frequency hearing loss secondary to hair loss in the organ of Corti are also common side-effects of cis-platinum (Pie! et al., 1974; Stadnicki et al., 1975; Higby et al., 1973; Talley et al., 1973). A rare allergic reaction consisting of oedema, wheezing, tachycardia and hypotension has also been described. All patients in whom this occurred had received cis-platinum without a hypersensitivity reaction prior to the allergic episode (Von Hoff et al., 1976b). Interactions between cis-platinum and other chemotherapeutic agents have been described in tissue culture (Drewinko et al., 1973) but more detailed investigations remain to be performed.

52


2. Antibiotics

lines, increased ability to degrade the drug appeared to be responsible for resistance (Miyak et al., 1975).

2. 1 Bleomycin 2.1.2 Pharmacokinetic Properties

Bleomycin is one of a number of antitumour antibiotics isolated from Streptomyces species. The commercially available compound is composed of a mixture of low molecular weight (1,500 daltons) peptides (Umezawa, 1974) and has demonstrated important antitumour activity in lymphomas, testicular carcinoma and various squamous cell carcinomas. The bleomycin peptides differ only in their terminal alkylamine group and can be separated by ion exchange chromatography. The most active antitumour peptide appears to be the A 2 species, which represents 50 % of the clinical preparation. The bleomycin peptides are degraded by aminopeptidases which are found in liver, kidney and malignant tissues, but which are not present in lung and skin (Umezawa et al., 1974), the primary normal tissues injured by bleomycin. 2.1.1 Mode ofAction The biochemical action of bleomycin has not been clearly elucidated, but these peptides have been shown to fragment DNA in vitro (Nagai et aI., 1969), yielding free thymine bases, and larger polynucleotide pieces. This fragmentation of DNA is enhanced by the presence of agents (such as dactinomycin) which intercalate into DNA (Bearden and Haidle, 1975), and by conditions which enhance superoxide or free radical generation (Ishida and Takahashi, 1975). Thus, synergistic activity is to be expected between bleomycin and doxorubicin (adriamycin) or dactinomycin (actinomycin D), since both the latter agents intercalate and may act as free radical transmitters. Cells are maximally sensitive to bleomycin when actively proliferating. During exposure to bleomycin, cells are delayed in their progression through the G 2 phase (intermitotic phase) of the cell cycle, thus undergoing synchronisation (Bearden and Haidle, 1975). Molecular mechanisms of resistance to bleomycin have not been identified in human tumours, but in at least two animal tumour

The clinical pharmacokinetics of bleomycin have only recently been studied (Broughton and Strong, 1976). Approximately 60 % of infused drug is excreted unchanged, while the remainder undergoes non-renal, and presumably metabolic degradation. By means of a radioimmunoassay which primarily measures the A 2 and B2 polypeptides, Holoye et al. (1977) determined that bleomycin disappears from plasma with at least 2 half-lives, the first having a value of 1.4 hours and the second 8.9 hours. Markedly prolonged elimination was observed in patients with compromised renal function, and was accompanied by increased host toxicity. On the basis of these findings, it is clear that extreme caution should be exercised in the use of bleomycin in patients concurrently receiving nephrotoxic agents such as high-dose methotrexate, cis-DD platinum, or aminoglycoside antibiotics or patients with compromised renal function. 2.1.3 Adverse Reactions

The primary clinical toxicity of bleomycin is manifest as cutaneous or pulmonary lesions. In both low and high dose (25mg/ rrr' or greater) intermittent schedules, cutaneous toxicity has been the .most common side-effect, although potentially lethal pulmonary toxicity is of more serious concern. Skin changes include induration, erythema, and tenderness of the digits, hands, elbows, in areas of previous radiation exposure. These changes may progress to desquamation and ulceration, and may recrudesce with later courses of other antineoplastic drugs. Pulmonary toxicity, while less frequent than skin lesions, has been the most serious side-effect leading to occasional fatalities. Persons over 70 years of age, those receiving total doses greater than 400mg or single doses greater than 25mg/ m 2, and those having prior or concomitant chest radiotherapy or underlying pulmonary disease have a greater risk of developing progressive drug-induced pneumonitis (Blum et

53


aI., 1973). Bleomycin-induced lung damage is unpredictably related to total dose in the individual patient, occurring in some cases after as little as 50mg total dosage. Symptoms of pulmonary toxicity are usually cough, dyspnoea and, at times, fever. Pleurisy may also be present. Physical examination may reveal little, if any auscultatory evidence of pulmonary infiltrates, in contrast to the patient's symptoms. In extreme cases cyanosis may be present. Radiological findings are often minimal despite severe symptoms, but later changes often include bibasilar infiltrates in an interstitial pattern with steady temporal progression. The gallium 67 lung scan is usually more clearly positive at early stages of bleomycin-induced lung toxicity, but does not distinguish this lesion from other types of diffuse lung disease found in this patient population, including infectious or malignant processes (Richman et aI., 1975). Open lung biopsy may be required to establish the diagnosis and should be undertaken at an early phase in evaluation of the patient. The underlying pathological lesion is composed of interstitial oedema, intra-alveolar hyaline membrane formation, hyperplasia of type II alveolar macrophages, and in later stages, collagen deposition. A model for bleomycin pulmonary toxicity has been developed in mice receiving twice-weekly doses for 4 weeks. The initial site of injury appears to be the intima of pulmonary arteries and veins, with later evolution of parenchymal changes as discussed previously (Adamson and Bowden, 1974). Pulmonary function tests have been of little value in predicting the onset of pulmonary toxicity, primarily because most patients undergoing bleomycin treatment show a progressive decrease in forced vital capacity, diffusion capacity, or both tests (Mosher et aI., 1972). At advanced stages of the pulmonary lesion, decreased arterial oxygen saturation, diffusion capacity, and vital capacity are seen. There is no known therapy for bleomycin lung toxicity, aside from discontinuation of the drug. Corticosteroids have been used with beneficialeffect in a few reported cases. Partial to full recovery of pulmonary function has been observed in our experience if patients can be

supported through the period of acute respiratory compromise. Because of experimental evidence that bleomycin has greatest cytotoxicity for cells in mitosis, clinical trials have been instituted using continuous drug infusion to provide continuous high drug levels as cells enter the sensitive DNA synthetic period. These infusions in doses of approximately 25mg/ day for 5 days, repeated every 4 to 5 weeks, have produced only occasional severe pulmonary toxicity in patients with testicular carcinoma, but have led to hypertensive episodes in 17 % of patients and hyperbilirubinaemia in 30 % (Samuels et al., 1975). These latter toxicities are rarely observed with conventional single dose injections. The drug interactions of bleomycin remain the subject for future investigation.

2.2 Anthracyclines The anthracycline antibiotics, adriamycin (doxorubicin) and daunomycin (daunorubicin), are two of the most important antitumour agents introduced into cancer chemotherapy within the last decade. These drugs are derived from fermentation products of the micro-organism Streptomyces peucetius var. caesius and differ only by the addition of an hydroxyl group that is attached to the alkyl side. chain of adriamycin as seen in figure 3 (Ghione, 1975). Both adriamycin and daunomycin are planar, unsaturated, tetracyclic compounds in which the C ring contains a quinone-type configuration and the A ring is bound to the amino sugar, daunosamine. Both drugs are remarkably effective anticancer agents; the clinical usefulness of daunomycin is principally in the treatment of acute leukaemias in adults whereas adriamycin has been employed effectively in a wide range of human tumours including breast and thyroid carcinomas, bone and soft tissue sarcomas, and Hodgkin's and non-Hodgkin's lymphomas (Carter, 1975). Because of the clinical utility of these drugs, a significant research effort has been mounted to develop


o

54

OH

daunorubicin (daunomycin): R = H doxorubicin (adriarnvcin): R = OH Fig. 3. The structures of adriamycin (doxorubicin) and daunomycin (daunorubicin).

new adriamycin-daunomycin analogues with an improved therapeutic index. Most attempts to date, including the conjugation of anthracyclines to antitumour antibodies, peptide derivatives, and to DNA have not improved the spectrum of activity or lessened the toxicity of the basic molecule (Levy et al., 1975; Wilson et al., 1976; Staquet et al., 1977). Structure-activity studies have revealed that both the tetracyclic chromophore and the daunosamine sugar must be present for activity and that the sugar moiety is required for DNA binding (Zunino et al., 1972). Furthermore, the stereochemistry of the sugar, and the position, availability and spatial distance of its amino group have all been found to have important effects on antitumour activity as well as toxicity (Adamson, 1974; DiMarco et al., ]976; Arcamone et al., 1976).

2.2./ Mode of Action The mechanism of action of the anthracyclines has been inferred from their ability to bind specifically to DNA by intercalation between adjacent base pairs, thus inhibiting DNA synthesis (Gabbay et al., I 976; DiMarco, 1975). Inhibition by adriamycin of DNA, RNA and protein synthesis has been documented in both cell-free systems and intact cells (Momparler et al., 1976). Furthermore, a recent study has shown that adriamycin will competitively and non-competitively inhibit DNA polymerase in vitro, and that the amino-sugar is critical for the ionic binding of anthracyclines to single-stranded DNA (Goodman et al., 1977). After administration, adriamycin and daunomycin quickly penetrate the cell where radiochemical data and intense fluorescence confirm nuclear uptake of


the drugs (DiMarco, 1975). It is therefore not surprising that adriamycin causes extensive degradation of DNA in vivo in many experimental systems (Lee and Byfield, 1976), acute fragmentation of rat liver and myocardial cell nucleoli (Merski et al., 1976), and chromosomal aberrations in human lymphoid and fibroblast cell lines treated with the drug (Newsome and Singh, 1977). A number of studies, most recently using flow microfluorometry, have indicated that the anthracyclines slow cell cycle progression and are cytotoxic in proportion to drug concentration and to the length of exposure of the cell line (Krishan and Frei, 1976; Clarkson and Humphrey, 1977). Exposure to adriamycin leads to an increase in late Sand G 2 phase cells with cells in S phase showing greatest sensitivity to cytocidal doses of the drug (Barlogie et al., 1976). The inhibition of cell proliferation by the anthracyclines may be completely explained by their intercalation into the DNA helix; however, some recent observations have suggested other mechanisms for the antimitotic and toxic effects of adriamycin and daunomycin. Highly specific binding of daunomycin to non-histone proteins from rat liver has been demonstrated; and thus, the anthracyclines may have a dual effect on- cell growth affecting the regulation of gene expression as well as the level of DNA synthesis (Kikuchi and Sato, 1976). When bound to closed circular DNA, reduced adriamycin and daunomycin cause a strand scission which is oxygen dependent and strongly inhibited by superoxide dismutase. Thus, the quinone function of the anthracyclines may be involved in free radical damage to DNA (Lown et al., 1977). These areas of active investigation may help in the future to explain the antitumour activity and unique toxic effects of adriamycin and daunomycin. 2.2.2 Pharmacokinetic Properties

After intravenous administration, adriamycin is rapidly distributed to the tissues. Though highly bound to plasma proteins, adriamycin concentrations in the heart, lung, liver and kidney of the rabbit were found to exceed plasma concentrations significantly for the 48 hours during which they were measured

55

(Harris and Gross, 1975). However, when measured in man, CSF levels of the drug were undetectable from 1 to I 8 hours after administration; and 6 patients previously responding systemically to adriamycin relapsed in the central nervous system while on therapy (Benjamin et al., 1974). The plasma disappearance of adriamycin and its metabolites has been described by Benjamin et al, (1977) and Bachur et ai. (1977). It consists of a triphasic pharmacokinetic pattern with a mean half-life of the first phase.of 12 minutes, of the second phase 3.3 hours and of the prolonged third phase 29.6 hours. Though extraction of the drug from the plasma occurs rapidly in the liver, adriamycin itself was found to be the most prominent species there. Metabolites appear rapidly in plasma and disappear according to biphasic. and triphasic patterns. Measurements of adriamycin disappearance by radioimmunoassay as compared with total fluorescence confirm the triphasic pattern but differ in the absolute concentration of drug in the plasma. The slow terminal excretion phase has provided a pharmacological rationale for the standard high dose intermittent therapy schedule (Creasey et al., 1976). The metabolism and disposition of the anthracyclines is still being defined. Adriamycin and daunomycin undergo extensive metabolism in the liver by both soluble and microsomal enzymes, as demonstrated by urinary metabolites (Takanashi and Bachur, 1976). The major changes include reductive or hydrolytic cleavage of the glycosidic group from the tetracyclic molecule; demethylation, sulphation or glucuronidation of the 4-hydroxyl group, and reduction of the alkyl side chain to adriamycinol and daunomycinol by an aldo-keto reductase. Only about 10 % of an administered dose of anthracycline is excreted in the urin~; the balance is thought to undergo biliary excretion, but its ultimate disposition in man has not been identified as yet (Benjamin, 1975a). Of the metabolites identified thus far, only the hydroxylation of the alkyl side chain is known to yield a compound with antitumour activity. In clinical use, changes in renal function appear to have little influence on elimination of the


anthracyclines, but hepatic dysfunction is known to delay drug disappearance and lead to severe toxicity in patients treated with full therapeutic doses (Benjamin, 1975a). A scheme for dose modification has been proposed which involves a 50 % reduction for bilirubin concentrations of 1.2 to 3.Omg / dl and a 75 % reduction for a bilirubin above 3mg/ dl. Adriamycin has usually been administered intravenously since less than 5 % of orally administered drug is absorbed (Harris and Gross, 1975). Recently, other modes of administration have been advocated. At least 2 groups (Haskell et al., 1975; Kraybill et al., 1977) have reported moderate success in the treatment of primary and metastatic tumours by intra-arterial infusion of adriamycin. The hazards and complications of indwelling catheters have been significant in these trials and would suggest judicious patient selection prior to intra-arterial therapy.

56

destruction of murine mammary carcinoma in vivo. Whether by altering pharmacokinetics or cellular transport, drug interactions involving adriamycin become more frequent as clinical use of the drug increases, is not yet clear. 2.2.4 Adverse Reactions

The clinical usefulness of anthracyclines is often limited by acute and chronic toxic side-effects. Most significant among the acute side-effects is depression of bone marrow function, with myelosuppression predominant (Benjamin et aI., 1974; Benjamin, 197 Sb). On occasion, thrombocytopenia can also be troublesome particularly in patients who have received prior radiation to the marrow, have bone marrow metastases, or who have compromised hepatic function (Benjamin, 197 Sb). Stomatitis is also common, particularly with regimens employing frequent low-dose administration (Bonadonna et al., 1972). Other acute side-effects reported with 2.2.3 Drug Interactions Drug interactions involving the anthracyclines anthracyclines include alopecia, nausea and vomiting have only recently begun to be appreciated. Such and rarely fever, chills, flushing and drowsiness (Bendiverse agents as phenobarbitone and amphotericin B jamin, 1975b). Adriamycin has been reported to actican effect adriamycin disposition. Phenobarbitone, vate the fibrinolytic system but the clinical signifithe classic inducer of hepatic microsomal drug cance of this finding is unknown (Bick et al., 1976). metabolising enzyme activity that also stimulates bili- Acute allergic reactions have been documented; they ary excretion, has been shown to increase adriamycin are usually of a localised cutaneous nature, although disappearance and decrease survival in" mice inocul- anaphylaxis has been known to occur (Benjamin, ated with L1210 leukaemia (Reich and Bachur, 1975b; Etcubanas and Wilbur, 1974). The most troublesome side-effect of long-term 1976). 4 of 7 patients with metastatic tumours previously resistant to adriamycin-containing combina- anthracycline administration is cardiotoxicity of 2 tion chemotherapy had objective responses when varieties: (1) ECG changes and arrhythmias, and (2) amphotericin B was added to the treatment pro- progressive cardiomyopathy which at autopsy reveals gramme. The mechanism of renewed response is fragmentation and drop-out of myofibrils, mitochondrial swelling and cytoplasmic inclusions. unexplained (Presant et al., 1977). Adriamycin interacts with heat. In tumour cells, ECG changes secondary to adriamycin and Hahn has described enhanced cytotoxicity if daunomycin include non-specific ST and T wave adriamycin is combined with 43°C temperatures changes, supraventricular tachycardias, and pre(Hahn et aI., 1975). However, hyperthermia mature atrial and ventricular contractions (Lenaz and prolonged greater than 30 minutes rendered the cells Page, 1976; l..efrak et al., 1973). These findings are insensitive to adriamycin, possibly via altered drug not dose-related, usually reverse several days to a transport (Hahn and Strande, 1976). Overgaard week after the last drug dose, and are rarely an indica(1976) has found that local hyperthermia combined tion to stop the use of the drug (Lenaz and Page, with systemic adriamycin markedly increases the 1976; l..efrak et aI., 1973). The cardiomyopathy asso-


ciated with anthracyclines may manifest itself clinically as the abrupt onset of congestive heart failure. Although the heart failure can be controlled by conventional treatment, death from this side-effect has been reported in 33 to 75 % of patients who develop it (Lenaz and Page, 1976; Lefrak et aI., 1973 ; Minow et aI., 1977). Clinical signs of the cardiomyopathy can present as late as 6 months after the last dose of daunomycin or adriamycin (Minow et aI., 1977; Von Hoff et aI., 1977b). A clear relationship exists between the cumulative dose of either drug and the incidence of congestive heart failure. Below a cumulative dose of 550mg/m 2 of adriamycin, the incidence of congestive heart failure is less than 1 % (Lenaz and Page, 1976; Lefrak et aI., 1973). The incidence rises sharply with doses above 550mg/m 2, affecting 30 % of patients so treated (Lenaz and Page, 1976; Lefrak et al., 1973). The risk of developing cardiomyopathy is increased in patients who have received mediastinal radiotherapy, concomitant cyclophosphamide, or who have uncontrolled hypertension or left ventricuIar strain (Minow et aI., 1977). Allowable cumulative doses of adriamycin should be lower than the usual 550mg/m 2 in patients with such risk factors. There are now reports that other drugs such as mithramycin and actinomycin D may also potentiate anthracycline cardiotoxicity (Kushner et aI., 1975). Clinical studies have been undertaken in the hope of predicting which patients are at high risk of developing cardiomyopathy. Patients who develop a greater than 30 % decrease in QRS voltage on ECG during adriamycin therapy were found to have a much greater risk of developing congestive heart failure. When this change is noted in a patient, the drug should be permanently discontinued (Minow et aI., 1977). Unfortunately, such changes are probably a late finding reflecting an already significantly damaged myocardium. Changes in systolic time interval and ejection fraction have been shown to occur early after the administration of adriamycin (Rinehart et aI., 1974; Ramos et aI., 1976). However, these tests may be too sensitive an indicator of ventricular

57

dysfunction to be a practical aid in determining when to stop therapy in an individual patient. Serial endomyocardial biopsies have shown progressive pathological changes in patients after anthracycline administration and have been used to adjust drug dose accordingly (Bristow et aI., 1977). Such a technique, while valuable, has limited practical usefulness. The various theories explaining cardiotoxicity have led to efforts to modify the anthracyclines or their mode of administration to avoid heart damage but retain antitumour effect. A low-dose weekly schedule of adriamycin at 30mg/m 2 has been reported to be unassociated with overt cardiac decompensation, despite cumulative doses in the range of 550 to 2500mg/m 2 (Weiss et aI., 1976). However, whether equivalent therapeutic activity results from this regimen is unknown. The theory that anthracycline cardiotoxicity may be related to specific sites on the molecule has led to the synthesis of many analogues (Adamson, 1974), some of which hold promise of being less toxic (Israel et aI., 1975). Other efforts have been directed toward selectively blocking uptake of the drug by cardiac tissue. Anthracyclines have been bound to DNA in the hope that the bulky complex would be taken up by tumour cells by pinocytosis but not by cardiac cells which have limited pinocytotic activity (Trouet et al., 1972). Nevertheless, studies in vivo have suggested that daunomycin is released from such complexes and is taken up by cardiac tissue. Thus, decrease in toxicity may not be related to differential uptake but rather to the complex functioning as a slow releaser of anthracyclines (Ohnuma et aI., 1975). The observation that strophanthin G may block myocardial uptake of adriamycin led to interest in pretreatment of patients with cardiac glycosides to prevent anthracycline heart toxicity (Arena et aI., 1972). However, two subsequent studies failed to confirm any decrease in cardiac uptake of adriamycin in the presence of cardiac glycosides (Bachur et aI., 1975; Smith and Kundi, 1976). Adriamycin has been linked covalently to tumour specific antibodies with the goal of directing the drug to target tumour tissue, sparing normal tissue (Levy et aI., 1975). Unfor-

58


tunately, such an approach may not be practical against most human tumours. Finally, the discovery that some cardiac changes induced by anthracyclines could be prevented by the addition of chelating agents to experimental systems has generated interest in the simultaneous use of anthracyclines and such antitumour agents as ICRF -159, which has chelating properties (Woodman et aI., 1975). Hyperpigmentation, ulceration, non-specific dermatitis and phlebosclerosis have all been described in humans treated with adriamycin or daunomycin (Benjamin, 1975b). Of particular interest is the additive effect of adriamycin and radiation therapy on the skin and oesophagus to produce severe dermatitis and oesophagitis (Greco et ale , 1976). Furthermore, adriamycin can activate dormant radiation dermatitis weeks after the cessation of radiotherapy, in a manner reminiscent of the action of actinomycin D (Benjamin, 1975b; Cassady et aI., 1975). There is experimental evidence that the action of radiation and anthracyclines is additive rather than synergistic; both induce similar breaks in DNA (Byfield et aI., 1977). With the increasing use of adriamycin in an adjuvant setting, another chronic toxicity may yet appear, the induction of second tumours. Anthracyclines have been shown to be mutagenic and

carcinogenic in both in vitro and in vivo systems (Philips et aI., 1975; Marquardt et aI., 1976).

3. Antimetabolites 3.1 Methotrexate One of the first antineoplastic agents, methotrexate, has continued to fmd an expanded range of clinical uses which now include the therapy of choriocarcinoma, maintenance and intrathecal therapy of acute lymphocytic leukaemia, pre- and post-operative therapy of head and neck cancer and treatment of breast cancer and osteogenic sarcoma in the adjuvant and metastatic setting. The clinical use of methotrexate has been considerably enhanced by improved understanding of its tissue toxicity as a function of drug concentration and disposition during conventional and high-dose therapy. It will be the object of this summary to review the pharmacological basis of antifolate therapy.

3.1.1 Mode ofAction The mechanism of drug action is shown in figure 4. Methotrexate, a 4 amino-I-deoxy-N'" methyl

Methotrexate N10-formyl FH4 5-10 N methenyl FH4

1 Dihydrofolate Reductase

•

----+ pUrine

nuc Ieo tid I es

/ DNA

~

N S - 10 methylene FH4

_

thymidylate

Leucovorin 5 (N -formyl FH4 ) Fig. 4. Metabolic pathways inhibited by methotrexate (MTX). Dihydrofolate (FH2 ) conversion to tetrahydrofolate (FH4 ) is blocked by MTX, leading to a deficiency of reduced folates required for both purine and thymidylate synthesis. This block may be bypassed by leucovorin (5-formyl tetrahydrofolate).


derivative of folic acid, is a potent inhibitor of the enzyme dihydrofolate reductase, with an inhibition constant in the range of 1 x 10 - 9M. Inhibition of this enzyme results in the accumulation of intracellular folates in their inactive dihydrofolate form, with consequent depletion of the tetrahydrofolate derivatives required for both purine and pyrimidine biosynthesis. Recent work supports the concept that an excess of free (unbound) methotrexate must be present in order to maintain complete inhibition of its target enzyme and ultimately DNA synthesis (Goldman et al., 1975). These findings correlate well with the experimental observation that free drug concentrations of I x 10- 8M or greater are required in plasma to maintain inhibition of DNA synthesis and to produce cytotoxicity in mouse bone marrow (Chabner and Young, 1973). Above this threshold level, cytotoxicity appears to be directly related to drug concentrations and to duration of tissue exposure (Pinedo and Chabner, 1977). The threshold concentration for cytotoxicity to murine gastrointestinal mucosa (5 x 10- 9M) is somewhat lower than that for bone marrow. The concentration required to kill tumour cells is uncertain and probably variable. Experimental evidenee suggests that insensitivity of human tumours to methotrexate is related to inability to transport the drug across the cell membrane, or alternatively to an increased concentration of the target enzyme, dihydrofolate reductase. This inference has provided the rationale for use of high doses of methotrexate, followed by the rescue agent, leucovorin (5-formyl tetrahydrofolic acid), in an effort to increase intracellular concentrations of free drug. Further rationale for the high dose regimens comes from the work of Zaharko et al. (1977). These workers have shown that bone marrow toxicity due to inhibition of thymidylate synthesis occurs at lower free drug concentrations than inhibition of purine synthesis, but the purine defect can be tolerated for only 18hrs before lethality supervenes, while thymidylate inhibition can be tolerated for greater than 48hrs (Zaharko et al., 1977). These findings may help explain the more rapid cell kill at high plasma concentrations.

59

Several approaches have been found to be effective in preventing toxicity of methotrexate. Leucovorin (citrovorum factor; calcium folinate), which replenishes the depleted tetrahydrofolate pool is able to prevent serious toxicity when given after otherwise lethal doses of methotrexate. Periods of high dose methotrexate infusion of up to 42 hours in duration have been used, followed by the antidote (Levitt et al., 1973). In theory, leucovorin should noncompetitively bypass the block in tetrahydrofolate synthesis, but effective rescue has been shown to require increasing doses of the folate in the presence of higher levels of methotrexate, probably due to competition for transport across the cell membrane (Pinedo et al., 1976). This principle is of considerable importance in managing patients with renal failure and delayed excretion of methotrexate, where small doses of leucovorin (15 to 25mg/ m 2) may be ineffective. Alternate approaches to rescue have been utilised in man, including nucleoside (thymidine) administration. In the studies of Ensminger et al. (1977), thymidine in doses of 8g/m 2 /day for 4 days was able to prevent serious toxicity due to methotrexate doses up to 6g/m 2 given as a 48 hour infusion. Longer periods of methotrexate infusion led to myelosuppression in 3 of 5 patients, including 1 toxic death. The trial of thymidine rescue is based on the possibility that normal tissues may be able to utilise thymidine more effectively than malignant cells, thus allowing exogenous thymidine to rescue the host, without reversing cytotoxic effects on the tumour. In tissue culture, a purine source as well as thymidine is required to prevent cytotoxicity. It is thought that circulating levels of purines in man may be sufficient to reverse this arm of the 2-pronged antifolate attack, and thus purines are not required for rescue clinically. With this background information, one can readily understand the otherwise complicated regimens of methotrexate therapy in current clinical use. In general, schedules are designed to maximise the concentration of methotrexate during a tolerable period of infusion of 6 to 42 hours in duration. The cytotoxic activity of residual methotrexate is then terminated by administration of leucovorin or thymi-


dine. The availability of sensitive assay methods for methotrexate has made it possible to design regimens which achieve specificblood levels for desired periods of time. These assay methods include a competitive protein binding method, in which bacterial or mammalian dihydrofolate reductase is used as the binding protein (Myers et aI., 1975), and a radioimmunoassay utilising rabbit antibody to methotrexate-albumin (Raso and Schreiber, 1975). Both methods appear to be relatively specific for methotrexate, although the contaminants in commercial drug preparations, and metabolites generated during drug administration have not been carefully evaluated for cross-reactivity in these assays.

60

unchanged drug. In general, methotrexate clearance parallels creatinine clearance (Kristensen et aI., 1975), although active renal tubular secretion of the antifolate is known to contribute to the elimination process. Third space fluid accumulations, such as ascites, act as reservoirs, may lead to prolonged elevations of plasma drug concentration and enhanced toxicity (Young et aI., 1976). In a similar manner, intrathecally administered methotrexate crosses slowly into the systemic circulation and leads to prolonged exposure of bone marrow and gastrointestinal mucosa; thus a given dose administered into the cerebrospinal fluid is potentially more toxic than the same dose given intravenously (Jacobs et aI., 1975). Conventional single doses (25 to 75mg / m 2) achieve peak concentrations in the range of 10- 5 to 3.1.2 Pharmacokinetic Properties 10-6M in plasma, while high-dose infusions (I to Methotrexate pharmacokinetics have been deter- 1Og/rn") lead to concentrations approaching 10-3M. mined for both conventional and high-dose regimens, As mentioned previously, human marrow appears and the half-lives of drug disappearance for both able to withstand concentrations above the cytotoxic types of therapy appear to be the same. Following an threshold of 10-8M for up to 42 hours with acceptainitial rapid phase of drug distribution, drug disap- ble toxicity, although higher concentrations are pears from plasma with an initial half-time of 2 to 3 tolerable for shorter periods of times. A number of hours, followed by a final phase of disappearance fatalities have resulted 'from high-dose therapy. In with a half-time of 8 to 10 hours (Stoller et aI., 1975), most instances this toxicity appears to have been the but when plasma disappearance was followed for a result of renal precipitation of methotrexate, with longer period of time, a third terminal elimination subsequent delay in drug excretion, and ineffective half-life of 6 to 69 hours (mean 27 hours) has been leucovorin rescue (Von Hoff et aI., 1977a). observed (Huffman et al., 1973). Radioactive Renal precipitation is believed to occur as a consemethotrexate cannot be used to accurately define quence of the high concentration and limited kinetics in this final phase of disappearance because solubility of the drug in urine. Attempts to force drug metabolites constitute the predominant species diuresis and alkalinise the urine with oral or of drug-derived material at these later time points intravenous bicarbonate have sharply reduced the (Kimelberg et al., 1977). The nature of these meta- incidence of drug toxicity in several reported series. bolites has not been established, except for the 7- Fluid and bicarbonate (3000ml/m 2/day) hydroxymethotrexate, a derivative of limited (120mEq/m 2/day), adequate to produce a urine outsolubility which may be of importance in the produc- put of 200ml/h at a pH of 7.0, are recommended by tion of renal failure during high-dose infusions many experienced therapists using methotrexate (Jacobs et al., 1976). doses above 1.5g/ m?/ day. In addition, monitoring of Oncologists observe considerable variability in the plasma methotrexate at 24 or 48 hours after the toxicity of methotrexate in patients receiving the beginning of the infusion is commonly practised since drug. This variability in most instances can be ex- it has been shown that high drug levels are predictive plained by differences in the rate of drug elimination, of an increased risk of serious toxicity (Nirenberg et which occurs predominantly via renal excretion of aI., 1977; Stoller et al., 1977). For example, in the


Jaffe regimen (Jaffe et al., 1974) used for adjuvant therapy of osteogenic sarcoma at the National Cancer Institute, toxicity was observed in our series of 395 infusions in only 7 patients, all of whom had 48 hour plasma levels greater than 9· 10 - 7M. Detection of an elevated drug concentration allows the physician to increase leucovorin dosage to the range of 1OOmg / m 2 and to institute other supportive measures as required by the patient's condition. In patients developing overt renal failure as a consequence of methotrexate renal toxicity, drug elimination is markedly prolonged, prompting attempts to promote drug clearance by peritoneal and haemodialysis. However, our experience suggests that unless the patient is completely anuric, haemodialysis makes little impact on drug clearance, while peritoneal dialysis appears to be of no value whatsoever. Alternative procedures to accelerate drug removal, such as enzymatic cleavage with bacterial carboxypeptidase and charcoal haemofiltration are currently under trial. Because of the limited solubility of methotrexate and the serious consequences which attend the development of renal failure, clinical trials have begun with aminopterin (2-4 diamino-pteroylglutamic acid), an analogue with greater potency. It may be possible to use this analogue in lower doses and obtain equal therapeutic effects.

3.1.3 Adverse Reactions Methotrexate is also the most commonly used agent for prophylaxis and treatment of carcinomatous and leukaemic meningitis. Minor side-effects such as arachnoiditis, nausea and vomiting occur in up to 30 % of patients receiving such therapy, while major complications including dementia, peripheral neuropathy, seizures, and coma afflict 10% of patients thus treated (Bleyer et al., 1973). These major complications are most common in adults receiving treatment for active disease, and usually appear after t to 2 weeks of treatment. Markedly elevated concentrations of methotrexate in the cerebrospinal fluid have been found in neurotoxic patients, indicating a defect in drug clearance as a possible

61

mechanism of toxicity. The half-life of methotrexate in spinal fluid of nontoxic patients is approximately t 2 hours. Limitation of the total dose to 12mg, regardless of patient body surface area, has been suggested in view of the higher frequency of complications in adults and the fact that cerebrospinal fluid volume changes little after the age of 2 years (Bleyer and Dedrick, t 977; Bleyer, 1977). Alternative approaches to the treatment of meningeal leukaemia are currently under investigation, including the use of small (1mg), frequent doses of methotrexate, administered through an Ommaya reservoir implanted subcutaneously and extending into the fourth ventricle. This form of therapy has the added advantage of bringing the drug directly to the intraventricular fluid, which receives limited drug exposure during intralumbar therapy because of poor drug distribution from the subarachnoid space to the ventricles (Shapiro et al., 1975). A second option under study is the use of systemic high-dose methotrexate. Several investigators have shown that cerebrospinal fluid levels are 3 to 10 % of those achieved in the systemic circulation and are probably cytocidal during the first 24 hours of systemic highdose infusion. The therapeutic efficacy of this approach has not as yet been established. In addition to renal and neurological toxicity, several other organ specific complications of methotrexate therapy have been described. Liver enzymes frequently rise immediately after high-dose infusions, but usually quickly return to pretreatment levels. A more serious lesion, portal fibrosis and cirrhosis, does complicate the long-term use of this drug for the treatment of recalcitrant psoriasis. Recent evidence has implicated a disturbance in choline synthesis (a folate dependent process) as the aetiology for a similar lesion in rats. Pulmonary infiltrates, accompanied by fever, have also occurred in rare cases, but usually resolve spontaneously and do not reappear with re-institution of the drug. Their aetiology is poorly understood. No specific therapy, other than temporary discontinuation of methotrexate, and lung biopsy if infiltrates progress, has been shown to be beneficial (Sostman et al., 1976).


62

Table I. Compounds possibly altering the pharmacokinetics or activity of methotrexate Drug

Effect on methotrexate (MTX)

Mechanism

Citrovorum factor

Reversal of action

Bypasses enzyme block

Sulphathiazole

Decreases absorption in mice

Destroys gut bacteria

Neomycin

Decreases absorption in mice

Destroys gut bacteria

Salicylates

May alter plasma level

Decreases renal clearance; displaces MTX from plasma proteins

Sulphonamides


Displaces MTX from plasma proteins

Phenytoin


Displaces MTX from plasma proteins

Vincristine

Increases intracellular level

Enhances MTX cellular uptake

Corticosteroids

Decreases intracellular level

Inhibits MTX cellular uptake

L-Asparaginase



Hydroxyurea



Bleomycin



Penicillin



Kanamycin



L-Asparaginase

Synergistic or antagonistic

Undefined

5- Fluorouracil


Undefined

Cytarabine


Undefined

Triamterene

'Pseudoresistance'

Increases dihydrofolate reductase level

Allopurinol

Decreases antitumour effect

Increases intracellular purine level

3.1.4 Drug Interactions

Several drugs have a potential to interact with methotrexate and their effects are summarised in table I. The effects of leucovorin have already been discussed above. Zaharko et al. (1969) investigated the effects of concurrently administered sulphathia-

zole or neomycin on methotrexate absorption in mice and found absorption to be inhibited. The mechanism for this effect, however, remains undefmed. Liegler et al. (1969) examined the effects of other drugs on methotrexate clearance in patients with acute leukaemia. Weak organic acids, such as salicylate,

63


reduced renal tubular clearance of methotrexate. As many antibiotics are salts of weak organic acids other such interactions might be expected to occur. An additional observation made by these investigators was that salicylate and sulphafurazole (sulfisoxazole) displaced methotrexate from plasma protein binding sites, an effect not well correlated with changes in renal clearance. Since decreased binding of a drug to serum albumin can increase the amount of free drug available at the peripheral receptor site (Koch-Weser and Sellers, 1976), an increased therapeutic effect as well as increased toxicity might be expected to occur, particularly if elimination is also inhibited. Plasma protein binding was also decreased by para-aminobenzoic acid, sulphonamides and salicylates according to another report (Dixon et al., 1965). Phenytoin (diphenylhydantoin) has also been said to increase methotrexate toxicity by displacement from binding to plasma proteins (Reilly, 1973). Agents which displace methotrexate from plasma protein binding sites, may increase the availability of methotrexate to tissue sites and might be expected to augment toxicity. As such, the concomitant administration of such drugs with methotrexate, when necessary, requires close clinical monitoring. Methotrexate enters mammalian cells by a carriermediated process and cellular uptake of drug may be affected by other drugs. Zager et aI. (1973) investigated the effects of several clinically useful antibiotics and antineoplastic agents on the intracellular accumulation of methotrexate and on its antitumour effects in LI210 leukaemia-bearing mice. Using doses felt to be estimates of achievable blood levels in man after usual single intravenous doses, corticosteroids, cephalothin, colaspase (Lasparaginasel and the vinca alkaloids were found to have the greatest effect on methotrexate uptake with lesser effects noted for hydroxyurea, bleomycin, penicillin and kanamycin. Hydrocortisone at 10- 5M and 10- 3M showed the greatest inhibition of methotrexate uptake and antagonised the antitumour effect of methotrexate in vivo, as measured by mean animal survival time, when hydrocortisone preceded methotrexate administration. Studies with human

leukaemia cells confirmed the inhibitory effects of cephalothin and hydrocortisone on methotrexate uptake (Bender et al., 1975). However, the clinical significance of these observations remains unclear. Other antitumour agents may also alter the activity of methotrexate. These interactions are dose-dependent and schedule-dependent and are well summarised by Capizzi et al. (1977). The potassium-sparing diuretic triamterene has been found to elevate levels of dihydrofolate reductase in human leukocytes when given as a diuretic (Roberts and Hall, 1968). As such, 'pseudoresistance' could be induced requiring increased amounts of methotrexate to achieve a therapeutic effect. The effects of allopurinol on the antitumour effects of methotrexate in L 1210 and P388 murine leukaemia have been investigated (Grindey and Moran, 1975). The concurrent administration of allopurinol partially reversed the antitumour effects of methotrexate against L 12 10 leukaemia when mean animal survival was measured, although efficacy against P388 leukaemia was not altered. Methotrexate growth-inhibitory effects on cells in tissue culture were not affected by allopurinol, nor was methotrexate toxicity altered by allopurinol as measured by acute toxicity studies. Further studies are required to better define this interaction and to clarify its clinical significance.

3.2 5-Fluorouracil After 20 years of clinical experience, 5fluorouracil continues to be widely used both as a single agent and, more recently, in combination chemotherapy against neoplasms of the gastrointestinal tract (Moertel, 1975), breast (Carter, 1972) and ovary (Young, 1975). Since the introduction of 5fluorouracil in 1957, several other fluorinated pyrimidines have also attracted clinical interest, but none has seriously challenged the parent compound for its position in cancer treatment. 5-Fluoro-2'deoxyuridine has superior activity against several murine tumours (Heidelberger et al., 1958), but does

64


OH

OH F

OH

F

F

FU

OH Ftorafur FUdR

Fig. 5. The structures of 5-fluorouracil (FU), fluoro-deoxyuridine (FUdR) and ftorafur.

not appear to be more effective than 5-fluorouracil against human tumours (Reitemeier et al., 1965; Moertel et al., 1967), probably because of its rapid conversion to 5-fluorouracil by the enzyme, nucleoside phosphorylase, which exists in a wide variety of tissues (Birnie et al., 1963). Ftorafur, a relatively new drug developed in the Soviet Union, is currently undergoing phase II evaluation in the United States, the preliminary results of which suggest a spectrum of activity similar to that of 5fluorouracil (Valdivieso et al., 1976). However, ftorafur appears to have less haematological toxicity in man (Smart et al., 1975; Valdivieso et al., 1976), a feature which may be exploitable in combination therapy with other myelosuppressive agents. Cohen ( 1975) has suggested that ftorafur may act as a depot form of 5-fluorouracil and that the liver is the primary site for this conversion. The slow release of 5fluorouracil in vivo could explain the milder bone marrow toxicity of ftorafur as continuous infusions of 5-fluorouracil are less toxic than an intravenous bolus dose (Seifert et al., 1975). The structures of these 3 agents are depicted in figure 5.

3.2.1 Mode ofAction Like other base and nucleoside antimetabolites, 5fluorouracil must be converted to its respective nucleotide in order to be active. Since cell membranes are impermeable to nucleotides, this activation process must occur within the target cell itself. With one important exception, both the anabolic and catabolic reactions of 5-fluorouracil are identical to those of uracil. As depicted in figure 6, 5-fluorouracil enters cells by passive diffusion (Jacquez, 1962) and is then converted to the nucleosides, fluorouridine and fluorodeoxyuridine, by nucleoside phosphorylase (Skold, 1958). Both fluorouridine and fluorodeoxyuridine are metabolised to their respective nucleotides, fluorouridine monophosphate and fluorodeoxyuridine monophosphate, by specific kinases (Chaudhuri et al. , 1958; Harbors et al., 1959). Alternatively, 5-fluorouracil may be converted directly to fluorouridine monophosphate by the action of a pyrimidine phosphoribosyltransferase (Reyes, 1969). Fluorouridine monophosphate can be incorporated into RNA where it may function as a false


messenger (Chaudhuri et al., 1958), or it can be converted to fluorodeoxyuridine monophosphate in a series of reactions which require ribonucleotide reductase (Kent and Heidelberger, 1972). It is well established that fluorodeoxyuridine monophosphate is an essentially irreversible inhibitor of the enzyme, thymidylate synthetase, which catalyses the formation of thymidylate from deoxyuridylate and, as a result, plays a crucial role in DNA synthesis (Cohen et al., 1958; Reyes and Heidelberger, 1965; Santi et al., 1974). The observations of unbalanced growth and thymineless death in bacteria following 5fluorouracil (Cohen et al., 1958) can be explained by this selective impairment of de novo thymidylate biosynthesis which would block DNA replication, but not RNA or protein synthesis (Heidelberger et al., 1960). The precise consequences resulting from 5fluorouracil alteration of RNA remain ill-defined, whereas the inhibition of DNA synthesis is better understood and is generally felt to be more important biologically (Heidelberger, 1974). Attempts to elucidate mechanisms of tumour cell resistance have focused on assays of activating and

dihydro

5-FU

!

65

degradative enzymes in the 5-fluorouracil pathway (Kessel et al., 1971; Kessel and Wodinsky, 1970; Skold et aI., 1962). Unfortunately, few investigations of this type have been carried out in human tumour tissue. A recent study of enzyme activity in human colon tumours does not include enough 5-fluorouracil treated patients to evaluate this approach in predicting tumour responsiveness (Nahas et al., 1974). Nevertheless, biochemical parameters associated with 5-fluorouracil response in human tumours have important therapeutic implications, especially for patients being considered for adjuvant therapy, and should be diligently pursued. 3.2.2 Pharmacokinetic Properties

Whereas the activation of 5-fluorouracil occurs in the target cell, inactivation occurs primarily in the liver (Chaudhuri et aI., 1958; Cooper et al., 1972). The pyrimidine ring is initially reduced by the ratelimiting enzyme, dihydrouracil dehydrogenase, and is subsequently cleaved nonenzymatically. The final products formed are o-fluoro-j-alanine, urea, ammonia, and carbon dioxide, which are primarily ex-

dihydrouracil dehydrogenase (I iver)

5-FUR uridine kinase

co , urea, F-J3 alanine 2

"'5-FUMP~ RNA

,

+

5-FUDP

5-FdUMP

Fig. 6. Pathways of metabolic disposition of 5-fluorouracil. 5-FU represents 5-fluorouracil, 5-FUR is 5-fluorouridine, 5-FUMP is 5-fluorouridylate, 5-FUDP is 5-fluorouridine diphosphate, and 5-FdUMP is 5-fluorodeoxyuridylate.


creted in the urine and lack antitumour activity. Earlier observations had suggested that the inability of certain tumours, including human breast and colon carcinomas, to degrade 5-fluorouracil was an important factor in their susceptibility to this agent (Mukherjee and Heidelberger, 1960; Mukherjee et al., 1963). However, inhibition of the catabolic pathway in rats did not lend support to this theory (Cooper et al., 1972). Following a single intravenous 15mg/kg dose of 5-fluorouracil, the drug rapidly diffuses into all body compartments and is distributed in a volume equivalent to the total body water (Chaudhuri et al., 1958). Peak plasma levels of 10- 4 to 10- 3M occur immediately and the drug is cleared rapidly from plasma with a half-life of 10 to 20 minutes. After 2 to 3 hours, plasma levels are below 10- 8M and are no longer detectable (Cohen et al., 1974; Finn and Sadee, 1975; Clarkson et al., 1965). Finn and Sadee (1975), using a highly sensitive mass fragmentography assay, were able to define a second phase of elimination in rats and in patients receiving oral 5-fluorouracii. The half-life for this phase was 20 hours. Approximately 20 % of the parent drug is excreted unchanged in the urine, most of this during the. first hour (Clarkson et al., 1965). The remaining 80 % of the administered dose is metabolised, primarily in the liver. The inactive metabolites formed are excreted in the urine over the next 3 to 4 hours with 90 % of the dose being accounted fer during the first 24 hours following administration (Mukherjee and Heidelberger, 1960; Clarkson et al., 1965). In contrast to the rapid extracellular clearance of the parent drug and its nucleosides, the active inhibitor, fluorodeoxyuridine monophosphate, has a much slower rate of disappearance from normal and tumorous murine tissues (Myers et al., 1975). More information about fluorodeoxyuridine monophosphate pharmacokinetics is needed to enhance our understanding of 5-fluorouracil action and toxicity. 5-Fluorouracil enters both malignant peritoneal and pleural effusions where it may persist for up to 12 to 24 hours (Clarkson et al., 1965). Intracavitary administration of 5-fluorouracil produces levels in the

66

effusion several hundred times higher than those obtained after systemic administration. Furthermore, plasma concentrations are 100 to 1,000 times lower than those in the effusion giving a wide margin of safety from adverse systemic effects (Clarkson et al., 1965). In spite of its limited lipid solubility, 5fluorouracil diffuses readily across the blood-brain barrier and distributes in cerebrospinal fluid and brain tissue (Bourke et al., 1973). Following 15mg/kg intravenously, maximum levels in the cerebrospinal fluid occur within 1 to 2 hours and persist at greater than 10- 8M for 12 hours (Clarkson et al., 1965; Bourke et al., 1973). A rapid rate of injection maximises brain and cerebrospinal fluid levels. Ftorafur, which possesses greater lipid solubility, has greater access to the central nervous system (Cohen, 1975), which explains its reported effectiveness against brain tumours and its higher incidence of neurotoxicity (Valdivieso et al., 1976). Oral administration of 5-fluorouracil produces widely variable plasma levels (Cohen et al., 1974; Finn and Sadee, 1975; Bruckner and Creasey, 1974; Hahn et al., 1975). Peak plasma concentrations are usually lower and occur-later than similar doses given intravenously. In addition, clearance of drug is prolonged. An explanation for these observations is found in the unreliable absorption of the drug, especially when it is given in an acidic solution, such as orange juice (Cohen et al., 1974). In spite of greater patient convenience, most studies have shown that the oral route has been associated with either fewer clinical responses (Stolinsky et al., 1975; Ansfield et al., 1977) or shorter durations of remission (Hahn et al., 1975). Sadee and Wong (1977) have reviewed the inter-relationship of the pharmacokinetics of 5fluorouracil with biochemical kinetics in monitoring therapy. 3.2.3 Dosage Schedule and Administration The optimum dosage and method of administering 5-fluorouracil has been debated for some time. The original 5 day loading course of Curreri et al. (1958) produced unacceptable toxicity. A modification of this regimen has recently been compared with weekly


intravenous 5-fluorouracil and an oral loading schedule in a randomised clinical trial (Ansfield et al., 1977). In this study, the modified intravenous schedule was found to be superior, particularly in patients with cancer of the colon and rectum. In other studies, single intravenous doses of 5-fluorouracil in excess of 20 to 30mg/kg have produced a disproportionate increase in toxicity (Jacobs et al., 1971; Horton et al., 1970). It is generally agreed that the 5 day continuous infusion produces considerably less haematological suppression, thus allowing higher doses to be given with safety (Hum and Bateman, 1975). However, it is doubtful that this schedule produces better therapeutic results than conventional schedules. The ineffectiveness of 5-fluorouracil in the treatment of patients with hepatic metastases has led to trials of hepatic artery infusion in the hope that high drug concentrations would be obtained in the hepatic parenchyma, while reducing systemic toxicity at the same time. Although a randomised clinical trial evaluating systemic and intrahepatic 5-fluorouracil has not been published, several reports have noted response rates of 35 to 85 % and some of these responses have endured for several years (Tanlon et al., 1973). Moreover, patients who have progressed on intravenous 5-fluorouracil may respond to intrahepatic administration (Buroker et al., 1976). The possible value of this therapy is offset by complications related to the indwelling catheter. However, intermittent courses by repeated percutaneous catheterisation may be a suitable alternative with minimal risk. Although the elimination of 5-fluorouracil has not been defined in patients with either renal or liver impairment, certain guidelines are available. As most of an administered dose is normally inactivated in the liver, 5-fluorouracil dosage may need to be modified in those patients with severe liver dysfunction. Severe renal disease may also require dosage modification, but is probably less critical. Ideally, such patients should be monitored with plasma drug levels to ensure optimum activity and to lessen the likelihood of toxicity.

67

3.2.4 Adverse Reactions

The more common toxicities of 5-fluorouracil relate to the bone marrow and gastrointestinal tract. Oral ulceration and diarrhoea tend to occur commonly with this agent and may be observed in about 20 % of patients. A knowledge of pyrimidine catabolism has provided an understanding of the cerebellar toxicity which occurs in 2 % of patients receiving 5fluorouracil and in 10 to 20 % of those receiving ftorafur (Koenig and Patel, 1970; Valdivieso et al., 1976). It has been proposed that some a-fluoro-~ alanine may be metabolised to fluoroacetate which is converted to fluorocitrate, an inhibitor of the Krebs cycle. Fluorocitrate produces neuronal lesions in cats similar to those produced by 5-fluorouracil (Koenig and Patel, 1970). 3.2.5 Drug Interactions

Little is known about the adverse drug interactions of 5-fluorouracil. Those interactions which have been described have evolved from attempts to affect the metabolism and biological activity of 5fluorouracil by simultaneously administering other agents. Interactions with prednisolone were investigated as a result of the frequent inclusion of corticosteroids into combination chemotherapy protocols, while phenobarbitone was chosen for study because of its ability to induce liver microsomal drug metabolising enzymes (Ambre and Fischer, 1971). No evidence for an effect of prednisolone or phenobarbitone pretreatment was observed on 5fluorouracil metabolite excretion, an expected observation since 5-fluorouracil metabolism is thought to be due to soluble rather than microsomal liver enzymes. When the effects of corticosteroids and vincristine on 5-fluorouracil activity in L 1210 cells were investigated, neither of these agents altered the ability of 5-fluorouracil to suppress de novo DNA synthesis, as measured by deoxyuridine incorporation. Similar results were obtained using human bone marrow cells (Bruckner et al., 1975). Other interactions, particularly between 5-fluorouracil and methotrexate, have already been discussed (section 3.1.4; table I).

68


3.3 Cytarabine (cytosine arabinoside) and Analogues 1-~-D-Arabinofuranosylcytosine (cytosine arabinoside, ara-C, cytarabine) is a synthetic nucleoside which differs from the naturally occurring nucleosides, cytidine and deoxycytidine, in a substitution of the sugar moiety, arabinose, for ribose or deoxyribose (fig. 7). Cytarabine was synthesised in 1959, found to be active against animal tumours in 1961 (Evans et al., 1961), and first used in clinical trials in 1964. The clinical usefulness of cytarabine is limited primarily to the treatment of leukaemia and lymphoma. The drug is the most active single agent in the treatment of acute myelogenous leukaemia, giving complete remission rates of 25 % when used alone and up to 50 % or more when used in combination with other agents such as daunomycin (daunorubicin), 6thioguanine, or alkylating agents (Kremer, 1975). Although its major use has been as an antineoplastic agent, cytarabrne has other in vivo and in vitro activities. It inhibits both T and B cell function, but has not been clinically used as an immunosuppressant

HO

OH

Cytidine

HO

Deoxycytidine

(Gray, 1973). Although cytarabine has shown in vitro antiviral activity, clinical studies have failed to demonstrate a beneficial effect of the drug in the treatment of herpes zoster or other viral infections when administered systemically (Stevens et al., 1973). Intrathecal administration of cytarabine has been useful in the treatment of meningeal leukaemia and lymphoma, particularly in patients resistant to methotrexate or in patients experiencing methotrexate-induced neurotoxicity (Band et al., 1973).

3.3.1 Mode qf Action The active form of cytarabine is cytosine arabinoside triphosphate, the intracellular levels of which correlate with the antitumour activity of the drug (Chou et al., 1975). Although cytosine arabinoside triphosphate is incorporated to a limited extent into both RNA (Chu, 1971) and DNA (Momparler, 1972), its primary mechanism of action is thought to result from inhibition of DNA polymerase (Rashbaum and Cozzarelli, 1976). As cytosine arabinoside triphosphate, like other nucleotides, does not cross cell membranes, the nucleoside, cytosine arabinoside (cytarabine), is used clinically and readily

HO

Cytarabine (cytosine arabinoside)

HO

OH

5-Azacytidine

Fig. 7. The structures of cytidine, deoxycytidine, cytarabine (cytosine arabinoside) and 5-azacytidine.

69


Ara-C _d_e_ox_y_cy_t_id_in_e_k_in_as_e_... Ara-CMP pyrimidine nucleoside monophosphate kinase cytidine deaminase

Ara-U (inactive)

nucleoside diphosphate kinase

deoxycytidine monophosphate deaminase

Ara-UMP (inactive)

dATP dGTP

TIP dCTP

-----1/;---. DNA DNA polymerase

Fig. 8. Pathways for intracellular metabolism of cytarabine (cytosine arabinoside; ara-C) to its active form, ara-CTP, and to its inactive deamination products, ara-U and ara-UMP.

enters cells by facilitated diffusion (Mulder and Harrap, 1975). Inside the cell, cytosine arabinoside undergoes a series of enzymatic phosphorylations, with eventual conversion to cytosine arabinoside triphosphate as shown in figure 8 (Momparler, 1974). Initial phosphorylation of cytosine arabinoside is accomplished by the enzyme, deoxycytidine kinase, which converts cytosine arabinoside to cytosine arabinoside monophosphate. This enzyme is present in low concentrations in leukaemic cells (Stoller et al., 1976) and is the rate-limiting step for activation of cytosine arabinoside in most circumstances. Cytosine arabinoside monophosphate is converted to cytosine arabinoside diphosphate by pyrimidine nucleoside monophosphate kinase (Hande and Chabner, 1976). A non-specific nucleoside diphosphate kinase converts cytosine arabinoside diphosphate to cytosine arabinoside triphosphate. Two degradative enzymes are present in high concentrations in human leukaemic cells, cytidine deaminase and deoxycytidylate deaminase which convert cytosine arabinoside and cytosine arabinoside monophosphate to inactive metabolites uridine arabinoside and uridine arabinoside monophosphate (fig. 8). Cytidine deaminase has been well studied and has a high affinity (low Km) for cytosine arabinoside

(Chabner et al., 1974). Little is known about cytosine arabinoside monophosphate deamination although relatively high concentrations of the inactive metabolite, uridine arabinoside monophosphate, have been found in leukaemic cells after cytosine arabinoside administration (Chou et al., 1975). Several mechanisms of resistance to cytarabine have been demonstrated in vitro: (1) decreased affinity of DNA polymerase for cytosine arabinoside triphosphate (Rashbaum and Cozzarelli, 1976); (2) an increase in intracellular pools of deoxycytidine triphosphate, the competitive substrate for DNApolymerase (Momparler et al., 1968); and (3) alterations in levels of the enzymes involved in cytosine arabinoside activation or degradation. Transport of cytosine arabinoside across cell membranes does not appear to be a determinant of sensitivity or resistance (Kessel et al., 1969). Major hopes for predicting resistance of leukaemic cells to cytarabine in man have been based on measuring changes in levels of activating enzymes (kinases) and degradating enzymes (deaminases). Tattersall et aI. (1974) has indicated that in some cases decreased levels of deoxycytidine kinase have been associated with tumour resistance, while Steuart and Burke (1971) have demonstrated that in some patients increased levels of the enzyme, cytidine deaminase, are


associated with development of cytarabine resistance. However, the pathways and controls of cytarabine metabolism are complex and intracellular levels of various enzymes vary greatly from patient to patient (Stoller et al., 1976). Therefore, measurement of intracellular levels of only certain activating and inactivating enzymes may be inadequate in predicting resistance. Indeed, in 2 clinical studies, measurement of intracellular levels of activating and inactivating enzymes in leukaemic cells has not been useful in determining sensitivity or resistance to cytarabine (Smyth et al., 1976; Chang et al., 1977). 3.3.2 Pharmacokinetic Properties The pharmacokinetics of cytarabine are largely determined by cytidine deaminase which is found in high quantities in the gastrointestinal tract, granulocytes and liver. The drug is inactive if given orally, due to rapid deamination in the gastrointestinal tract and liver. Following intravenous administration, there is rapid disappearance of cytarabine with an initial distribution half-life of 7 to I 7 minutes and a second or elimination phase half-life of 30 to 200 minutes. 80 % of the drug is recovered in the urine within 24 hours, 72 % being the inactive metabolite, uridine arabinoside (Ho and Frei, 1971). Cytarabine crosses the blood-brain barrier to some extent and cerebrospinal fluid cytarabine levels of about 40 % of plasma levels can be achieved (Ho and Frei, 1971). Intrathecal administration of cytarabine achieves much higher cerebrospinal fluid levels. The half-life of cytarabine in the cerebrospinal fluid (tt /2 of 2 hours) is prolonged over that in plasma due to absence of cytidine deaminase in the cerebrospinal fluid, resulting in slower conversion of cytosine arabinoside to uridine arabinoside. Since cytarabine is rapidly inactivated, continuous infusions or multiple repeated doses are needed to produce cytotoxicity in vivo. Single doses of up to 2g/m 2 produce no toxicity, whereas doses of l g/rn? produce severe myelosuppression if given by continuous infusion of 48 to 96 hours (Frei et aI., 1969). To circumvent the need for long continuous infusions of cytarabine, alternative drugs have been used.

70

Cyclocytidine (fig. 9), another cytidine derivative, is slowly hydrolysed to produce cytosine arabinoside and thus acts as a sustained release form of cytarabine. However,' recent clinical trials have indicated that cyclocytidine produces parotid pain and postural hypotension in addition to the usual toxicities seen with cytarabine, findings which may well limit its clinical usefulness (Burgess et al., 1977). Another method of prolonging the effect of a single bolus of cytarabine has been the concomitant use of tetrahydrouridine (fig. 9), a potent inhibitor of cytidine deaminase. Clinical trials presently under way indicate that the concomitant use of tetrahydrouridine prolongs the plasma half-life of cytarabine and that the dose of cytarabine as well as the frequency of administration can be decreased (Kreis et aI., 1977). 3.3.3 Adverse Reactions Common toxicities seen with the use of cytarabine include bone marrow suppression, nausea and vomiting. The nausea and vomiting are usually not severe and can be controlled with antiemetics. The incidence and degree of myelosuppression are dependent on dose and duration of administration. Large single doses of up to 2g/m 2 produce minimal effects, as do continuous infusions below I Omg/ m 2 / day (Ellison et al., 1968). However, intravenous doses of I to 3mg/kg/day for 5 to 10 days produce myelosuppression, anaemia and thrombocytopenia in most patients (Kremer, 1975; Bodey et al., 1969). The median white cell nadir is seen on day 8 with a median recov-. ery time of 15 days. Alopecia, mucosal ulceration, and diarrhoea are other side-effects but are relatively uncommon. Transient elevations of hepatic transaminases (1.5 to 4 x normal) have been noted in 10 to 20 % of patients. These elevations appear dose related, return to normal within 1 week, and are not a cause for cessation of therapy (Bodey et al., 1969). Occasional idiosyncratic reactions to cytarabine have been noted. Intrathecal administration of cytarabine may rarely produce arachnoiditis and other neurological dysfunction (Band et aI., 1973).

71


HO

H

::l~~ HO Cyclocytidine

HO

OH

Tetrahydrouridine (THU)

Fig. 9. The structures of cyclocytidine and tetrahydrouridine.

Dose alterations are not needed for renal failure as the drug is primarily inactivated by hepatic deamination. Dose modifications for patients with severe hepatic dysfunction are probably needed to compensate for decreased hepatic deamination, although the precise dose schedule has not been established.

3.3.4 Drug Interactions Few interactions have been described for this class of drug. The only one of significance involves cytarabine and methotrexate and has been described in section 3.1.4 (table I).

3.4 5-Azacytidine 5-Azacytidine is a cytidine analogue presently under investigational study. The drug was synthesised in 1964, noted to have antineoplastic activity in 1966, and has been undergoing clinical trials in the United States since 1970. Its structure is illustrated in figure 7. Initial clinical trials have demonstrated activity against leukaemia (Von Hoff et al., 1976a; Vogler et al., 1976) but little activity against solid tumours (Quagliana et al., 1977).

3.4.1 Modeo.fAction The biochemical pharmacology of this drug is complex. Like cytarabine, 5-azacytidine is most active during the S-phase of the cell cycle (Li et al., 1970b). The intracellular metabolism of the drug is very much like that of cytarabine except that the initial phosphorylating enzyme is uridine-cytidine kinase instead of deoxycytidine kinase. Uridine-cytidine kinase is present in low quantities within leukaemic cells and has a relatively low affinity for 5-azacytidine and, therefore, may well be rate-limiting in 5-azacytidine activation. 5-Azacytidine monophosphate is phosphorylated to 5-azacytidine diphosphate which is then converted to 5-azadeoxycytidine diphosphate by ribonucleotide reductase. 5-Azadeoxycytidine diphosphate is phosphorylated to 5-azadeoxycytidine triphosphate and subsequently incorporated into DNA (Li et al., 1970a). 5-Azacytidine is also phosphorylated to 5azacytidine triphosphate which inhibits RNA synthesis (Li et al., 1970a). Since 5-azacytidine triphosphate is a weak inhibitor of RNA polymerase (Lee and Momparler, 1977), the primary mechanism of action is probably through incorporation of unstable nucleotides into nucleic acids with subsequent dis-

72


ruption of the secondary structure of these molecules leading to misreadings with defective protein synthesis (Cihak et al. , 1967) and degradation of polyribosomes (Levitan and Webb, 1969). One mechanism of in vitro resistance to 5azacytidine has been through deletion of the activating enzyme, uri dine-cytidine kinase (Vesely et aI., 1970). Since the initial activating enzymes of cytarabine and 5-azacytidine are different, cells resistant to cytarabine by means of deletion of deoxycytidine kinase should still be sensitive to 5azacytidine. 3.4.2 Pharmacokinetic Properties 5-Azacytidine undergoes spontaneous decomposition in alkali or neutral solutions and decomposes in water when stored at 40°C with a half-life of 4.4 hours. However, when 5-azacytidine is made up in Ringer's lactate (pH 6.2) at 25°C the half-life is increased to 65 hours and to 94 hours at 20°C. 5-Azacytidine undergoes rapid metabolism following a single intravenous injection with less than 2 % of the administered dose remaining in plasma as 5-azacytidine after 30 minutes (Isralli et aI., 1976). After a single injection of 14C-Iabelled 5-azacytidine, 40 % of the radioactivity is excreted in the urine within 4 hours and 90 % within 24 hours, mostly as metabolic products. In contrast to a single bolus dose, plasma levels of 5-azacytidine are 13 % of the total plasma radioactivity following continuous infusion of 14C-Iabelled 5-azacytidine. 5-Azacytidine is also well absorbed following subcutaneous administration (Troetel et aI., 1972). 3.4.3 Adverse Reactions Major toxicities to 5-azacytidine have included severe nausea and vomiting which have been present in over 70 % of patients following bolus injection and have been a major cause of patient refusal to receive subsequent doses (Von Hoff et aI., 1976a; Vogler et aI., 1976; Quagliana et aI., 1977). The drug has usually been administered rapidly because of the previously mentioned instability in solution. However, if the drug is made up in Ringer's lactate and kept at

temperatures lower than 25°C, less than 10% of the drug decomposes over 8 hours and continuous infusions of 5-azacytidine may be given by mixing up new drug every 6 to 8 hours (Isralli et al., 1976). A marked reduction in gastrointestinal toxicity (without loss of antitumour activity) is noted when the drug is administered continuously rather than as an intravenous bolus (Vogler et al., 1976). The major dose limiting toxicities of 5-azacytidine have been leukopenia and thrombocytopenia which occur in the majority of patients. The white count nadir is seen on days 14 to 17 following a 5-day infusion. Other infrequent toxicities associated with 5-azacytidine have included abnormalities in liver function tests, myalgias, stomatitis, rash and transient temperature elevations (Von Hoff et aI., 1976a; Bellet et aI., 1973; McCredie et al., 1973; Shindler et al., 1976).

4. Plant A lkaloids and Epipodophyllotoxins 4.1 Plant Alkaloids The vinca alkaloids are derived from the periwinkle plant (Vinca rosea) and were first recognised to have antitumour properties in the 1960's (Johnson, 1963). Vincristine and vinblastine were the 2 original agents entered into clinical use (Karon et aI., 1962; Hodes et aI., 1960; Carter and Livingston, 1976). Recently, new analogues, particularly vindesine, have entered phase I clinical trials (Blum and Dawson, 1976). The structures of these compounds are illustrated in figure 10. 4.1.1 Mode ofAction All 3 drugs are dimeric alkaloids composed ofvindoline and catharanthine which, by themselves, are devoid of antitumour activity. The antitumour effects of the dimeric alkaloids appear to derive from their ability to rapidly and quantitatively enter cells (Bleyer et al., 1975) and bind to tubulin (Owellen et aI., 1972) with a high affinity (K m = 10 - 6M). Binding to microtubular components tends to disrupt processes that require a functionally intact microtubular system

73


OH

OCOCH 3

17

OH COOCH 3 Vinblastine R = CH3 Vincristine R = CHO

at-----------------------------------------~ OH

N

17

OH

I

CH 3 OH

CONH

2

ba..-

----J

Fig. 10. The structures of the vinca alkaloids, vinblastine (a), vincristine (a), and vindesine (b).

such as mitosis, maintenance of cell shape, and mobility of cell surface components. In addition, vincristine has been shown to inhibit DNA synthesis in vivo and both drugs, vincristine and vinblastine, have been shown to inhibit RNA and protein synthesis in vitro (Creasey and Markiw, 1965; Creasey, 1968). However, as these latter effects require pharmacological drug concentrations, their clinical relevance is questionable.

4. J.2 Pharmacokinetic Properties The study of the absorption, distribution and excretion of vinblastine and vincristine has been facilitated by the recent synthesis of tritiated radiochemicals. Human studies (Owellen and Hartke, 1975; Owellen et al., r977a) with vinblastine are largely consistent with previous work in dogs (Creasey et al., 1975) and suggest that the clearance of drug-derived


radioactivity from the blood is biphasic with initial distribution and terminal (elimination) half-lives of = 4.5 minutes and = 190 minutes, respectively. Extensive binding of vinblastine to formed blood elements occurs, with platelets binding more extensively than red cells or leucocytes. During a 72 hour period of study, = 21 % of drug appeared in the urine as unchanged vinblastine. The extensive retention of radiolabel (= 46 % at 72 hours), and suggestion of hepatic metabolism based on selective alteration of the faecally excreted vinblastine, are noteworthy. Similar studies have been performed using [3H]vincristine in both animals (Castle et al., 1976) and in man (Bender et al., 1977; Dwellen et al., 1977b). The human pharmacokinetics are characterised by a triphasic decay of blood radioactivity with half-lives of 0.85, 7.4, and 164 minutes, respectively, according to one group (Bender et al., 1977) and a biphasic decay with half-lives of 3.4 and 155 minutes, respectively, according to the other (Owellen et al., 1977b). Again, extensive binding of vincristine to formed blood elements was noted, with > 50 % bound 20 minutes following intravenous injection. Over a 72 hour period of study, = 12 % of radio label was excreted in the urine with about one-half of this as metabolites, and = 69 % was excreted in the faeces with = 40 % as metabolites. The extensive faecal excretion of drug and significant metabolism of drug excreted by this route suggests that biliary excretion and metabolism are important in man, similar to what has already been described in animals (Castle et al., 1976). Preliminary studies with Vindesine (Nelson et al., 1976; Owellen et al., 1977b) suggest similar pharmacokinetics to vinblastine and vincristine but more detailed disposition studies remain to be performed. The biliary elimination of the vinca alkaloids has prompted clinicians to empirically modify drug doses in the face of hepatic compromise, but no rigid guidelines exist. 4.1.3 Adverse Reactions The toxicities of vincristine and vinblastine are strikingly different considering the close structural

74

similarity of the 2 compounds. The dose-limiting toxicity of vinblastine is leucopenia with a nadir occurring 4 to 7 days post-administration (Livingston and Carter, 1970). Thrombocytopenia and anaemia may also occur, but less frequently. Although neurological toxicity may be produced by any agents of the vinca alkaloid family, it is especially characteristic of vincristine as seen by loss of deep tendon reflexes, paraesthesiae, peripheral neuropathy and cranial nerve findings (Weiss et al., 1974; Rosenthal and Kaufman, 1974). This unique toxicity may accrue from the high concentration of microtubular protein in neural tissue but does not explain the differences between vincristine and vinblastine with respect to neurotoxicity as their association constants (Ka) for tubulin are 8.0 and 6.0 Iitres/rnole, respectively (Owellen et al., 1972). Similarly, as both compounds bind avidly to formed blood elements, the greater myelotoxicity of vinblastine is unexplained. 4.1.4 Drug Interactions The interactions of vincristine and vinblastine with other drugs is of interest although their clinical significance remains to be defined. Low concentrations (0.005mg/mO of several amino acids, namely glutamic acid, aspartic acid, ornithine, citrulline and arginine, completely reverse the cytotoxic effect of vinblastine in tissue culture (Johnson et al., 1960). This observation may have clinical relevance for the patient receiving intravenous hyperalimentation. Vincristine has been shown to augment methotrexate uptake by human acute myeloblastic leukaemia cells in vitro at a clinically achievable concentration of 0.1 pM (Bender et al., 1975). However, more recent work on human lymphoblastoid cells in tissue culture demonstrates no augmentation of methotrexate uptake until 100-fold higher vincristine concentrations are reached (Warren et al., 1977). This disparity raises questions regarding individual tumour thresholds for this effect and minimises its clinical utility at this time. Similar effects have been described for vinblastine in murine L 12 10 leukaemia cells in vitro (Zager et al., 1973).

75


4.1.5 New Plant Alkaloids A new plant alkaloid, maytansine, first isolated and described in 1972 (Kupchan et al., 1972), has recently entered phase I clinical trials (Chabner et al., 1972). Its structure is depicted in figure II. The drug appears to act in a similar manner to the vinca alkaloids (Remillard et al., 1975; Wolpert-DeFilippes, I 97 Sa.b) and also avidly binds to tubulin, with evidence for its sharing a common binding site with both vincristine (Mandelbaum-Shavit, 1976) and vinblastine (Bhattacharyya and Wolff, 1977). Its human pharmacology remains to be investigated.

4.2 Epipodophyllotoxins The epipodophyllotoxins, epipodophyllotoxin ethylidene (VP-16) and epipodophyllotoxin thenylidene (VM-26), are derived from the roots and rhizomes of the mayapple or mandrake plant. Their antitumour effect was recognised as early as 1946

(King and Sullivan, I 946) but their structures remained elusive until 1971 (Keller-Juslen et al., 1971). They are depicted in figure I 2. Their clinical activity, particularly in leukaemia and lymphoma (EORTC, I 972, 1973; Rozencweig et al., 1977) was recognised early and the agents are now undergoing extensive phase III investigation in both the United States and in Europe. 4.2.1 ModeQfAction Both agents appear to act as mitotic spindle poisons, initially producing a metaphase arrest which is later superseded by preventing cells from entering mitosis by acting in the G 2 phase of the cell cycle (Stahelin, 1970, 1973). Moreover, cells blocked by VP-16 or VM-26 are unable to resume cell cycle traverse when these drugs are removed from the bathing medium in vitro (Krishan et al., 1975). Recent work on VP-16 in HeLa cells suggest that IOO}JM concentrations of drug do not inhibit the in vitro assembly of microtubules but do inhibit thymidine and

o

CL

o

I

I

I

Fig. 11. The structure of maytansine.

CH3

0

II I II O-C-CH---N-C-CH 3 :I I CH3


76

OH

OH

(0

(0

0

0

/0-CH CH3-CH

~O

/O-CH

0

2

S

OH

OH

0

2

CH

C(~O OH

VP·16

VM·26

Fig. 12. The structures of the epipodophyllotoxins, VP-16 and VM-26.

uridine uptake (Loike and Horwitz, 1976a). Further, VP-16 induces single-stranded DNA breaks in the same cell line (Loike and Horwitz, 1976b).

4.2.2 Pharmacokinetic Properties The pharmacokinetics in man of both agents have been studied by Creaven and Allen (I 975a,b). Both agents are highly water-insoluble and can be given intravenously only by being solubilised in a detergent mixture. VP-16 has a biphasic plasma decay with initial distribution and terminal elimination half-life values of = 2 hours and = 13.5 hours, respectively. The drug is distributed into 32 % of the body water and has poor penetration into the cerebrospinal fluid. Over a 72 hour period, < 16 % of drug is excreted in the faeces and 43.5 % is excreted in the urine, of

which 66.8 % represents parent compound. The fate of the retained fraction (= 40 % of the administered dose) remains undetermined. VM -26 is characterised by a triphase plasma decay with a long terminal elimination phase half-life of 11 to 38.5 hours. Cerebrospinal fluid penetration is poor with a 100-fold difference between cerebrospinal fluid and plasma levels at 24 hours. This may be due to the extensive protein binding C> 90 %) of VM-26. Over a 72 hour period, < 10% of drug is excreted in the faeces and 45 % is excreted in the urine of which 79 % represents metabolites, in contrast to VP-16. Instantaneous mean peak blood levels of VM-26 and VP -I 6, given at maximally tolerated single intravenous doses of 67mg / m 2 (Muggia et al., 1971) and 290mg/m 2 (Creaven et al., 1974), are 14mg/ml and 29mg/ml, respectively. The difference between


the maximally tolerated doses of both drugs appears to correlate with their pharmacokinetics, as the renal clearance of VP-16 is 6-fold greater than that for VM-26 (Allen and Creaven, 1975). However, as the renal clearance for both drugs is low « 13.6ml/min) augmented toxicity in patients with impaired renal function is not likely. Further, while the role of hepatic compromise in altering toxicity is unknown, the low faecal excretion of both drugs suggests that hepatic function does not playa major role in altering toxicity. 4.2.3 Adverse Reactions The toxicities of the epipodophyllotoxins are several. Nausea and vomiting are experienced by 25 % of patients, whether the drug is given intravenously (Creaven et al., 1974; Nissen et al., 1972) or orally (Nissen et al., 1976). Leucopenia (WBC < 5,000/mm 3) was seen in 100% of patients given weekly intravenous doses of VP-16 of 170mg/ m", with thrombocytopenia (platelets < 100,000/mm 3 ) occurring in 13% of patients (Creaven et al., 1974). Other schedules, such as 50mg/m 2 daily for 5 days, produced leucopenia in only 16 % of patients and thrombocytopenia in only 7 % (EORTC, 1973). The schedule and dose dependence of myelosuppression is true for VM-26, as well. Alopecia and stomatitis has also been reported with the epipodophyllotoxins. A unique toxicity of these agents appears to be related to the vehicle used for their intravenous administration. Transient episodes of hypotension have been reported during the period of drug administration which respond rapidly to either slowing the infusion rate or to temporarily interrupting drug administration. Drug interactions have not been described for the epipodophyllotoxins to date.

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Author's address: Dr Richard A. Bender, Medicine Branch and Clinical Pharmacology Branch, National Cancer Institute, Building 10, Room 12N226, 9000 Rockville Pike, Bethesda, Maryland 20014 (USA).

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