Pharmacokinetic study of cystemustine, administered on a weekly schedule in cancer patients

Annals of Oncology 13: 760–769, 2002 DOI: 10.1093/annonc/mdf098

Original article

Pharmacokinetic study of cystemustine, administered on a weekly schedule in cancer patients E. Cellarier1,2*, C. Terret3, P. Labarre2, R. Ouabdesselam1, H. Curé1, C. Marchenay1,2, J. C. Maurizis2, J. C. Madelmont2, P. Chollet1,2 & J. P. Armand3 1

Centre Jean Perrin and 2INSERM U484 Clermont-Ferrand; 3Institut Gustave Roussy, Villejuif, France

Background: Cystemustine is a chloroethylnitrosourea mostly active in humans against glioma and melanoma. The present report describes the results of a new phase I trial with cystemustine administered on a weekly schedule. The pharmacokinetic and pharmacodynamic properties of cystemustine were investigated. Patients and methods: Forty-three patients entered this study. Cystemustine was administered at dose levels ranging from 30 to 60 mg/m2. The drug was given on days 1, 8, 15 and 22, followed by a 4-week rest period. Results: Thrombocytopenia was the dose-limiting toxicity and appeared to be reversible, but probably cumulative. This toxicity appeared dose-related, both in frequency and severity. The maximum tolerated dose was 60 mg/m2. Nonhematological toxicity was generally mild. Three partial responses were observed at dose levels of 50 and 60 mg/m2. Pharmacokinetics analysis showed mono- or biphasic cystemustine blood disposition with a mean α half-life of 4 min and mean terminal half-life of 49 min. Conclusions: There was a clear linear relationship between the area under the blood drug concentration–time curve (AUC) and the dose of cystemustine (P <0.001). There was also a significant relationship between the AUC and the toxic effects of cystemustine on platelets, granulocytes and leukocytes (P <0.001). A reasonable starting dose for phase II studies is 40 mg/m2, with dose escalation based on blood cell counts. Key words: chloroethylnitrosourea, cystemustine, hematotoxicity, pharmacokinetic– pharmacodynamic relationships, phase I

Introduction Chloroethylnitrosourea (CENU) compounds are highly active cytotoxic drugs against a variety of hematological and solid tumors. However, their clinical usefulness has been limited by non-selective host toxicity, particularly myelosuppression [1]. The chemistry of these compounds have been extensively studied [2, 3] and a variety of structural modifications have been tested, aimed at optimizing their activity against murine leukemia and/or decreasing their hematological toxicity. Cystemustine {N′-(2-chloroethyl)-N-[2-(methyl sulphonyl) ethyl]-N′-nitrosourea} is a new CENU derived from 2-chloroethylnitroso-carbamoylcysteamine (CCNC), an isomeric mixture of cysteamine (Figure 1). This new compound presented very promising anticancer properties against a variety of

*Correspondence to: Dr E. Cellarier, Unité d’Oncothérapie Appliquée, Center Jean Perrin, 58 Rue Montalembert, BP 392, 63011 Clermont-Ferrand, Cedex 1, France. Tel: +33-4-7327-8005; Fax: +33-4-7327-8029; E-mail: [email protected] © 2002 European Society for Medical Oncology

murine tumors [4, 5]. Based on survival data and number of cures, it demonstrated, for solid tumors, an equivalent and often better chemotherapeutic index than the CENU analogs currently in clinical use, i.e. carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU) and fotemustine [6, 7]. Furthermore, interest in cystemustine as a clinically active agent focuses on its high alkylating activity [8] and its favorable solubility characteristics. The logarithm of P, the octanol/ water partition coefficient of cystemustine, is 0. This value suggests that this agent readily diffuses across cell membranes and the blood–brain barrier. On this basis, cystemustine was selected for clinical development. A first phase I trial was conducted in an intrapatient escalation schedule, which demonstrated that the doselimiting toxicity of cystemustine is hematological, mainly thrombocytopenia and leucopenia [9]. For phase II trials, the safe dose was 60 mg/m2 (then increased to 90 mg/m2) every 2 weeks. It must be noted that during phase I–II evaluation, some objective responses were obtained against gliomas,

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Received 8 March 2001; revised 1 October 2001; accepted 23 October 2001

761

Figure 1. Structural formula of cystemustine, N′-(chloro-2 ethyl) N-[(methyl sulfonyl)-2 ethyl] N′-nitrosourea.

Patients and methods Patient selection Selected patients with histologically confirmed advanced malignancy, but without standard alternative treatments, were enrolled on to this phase I clinical trial of cystemustine. The requirements for entry were an expected survival of at least 3 months; a World Health Organization (WHO) performance status (PS) <2; aged 18–75 years. No chemotherapy or extensive radiotherapy was administered 4 weeks before cystemustine therapy (prior nitrosourea or mitomycin-C treatments were a criteria of exclusion). The minimal hematological parameters required were leukocytes >4000/µl; platelet count >100 000/µl and hemoglobin >10 g/dl. Adequate renal and hepatic functions were also required (creatinine <140 µmol/l; bilirubin <30 µmol/l and AST, ALT, γ-GT <3× upper normal limit). Patients with a history of hemorrhagic risk (gastric ulcers, cirrhosis, coagulopathy) or neuropsychological problems were excluded, as well as patients with uncontrolled infection. Written informed consent was obtained from all participants, and the protocol was reviewed by the Auvergne Ethics Committee (CCPPRB).

Drug formulation and administration Cystemustine was synthesized in the INSERM U484 laboratory, Clermont-Ferrand, France. Lyophilization and sterilization for the

Dose escalation The starting dose was 30 mg/m2/week for 4 consecutive weeks. This dose (120 mg/m2 cumulated for 4 weeks) represented 67% of the recommended dose of the previous phase II study using 180 mg/m2/4 weeks, separated in two injections of 90 mg/m2. A linear increase was chosen in the previous therapeutic domain of cystemustine, with increments of 10 mg/m2: dose levels were 30, 40, 50 and 60 mg/m2. A minimum of three assessable patients were treated at every dose level, and no intrapatient dose escalations were allowed. The maximum tolerated dose (MTD) was defined as the dose resulting in severe toxicities (grade 4 for >5 days neutropenia; grade 4 thrombocytopenia or grade 3 non-hematological toxicity except alopecia and nausea/vomiting) in 50% or more of the patients. It was then decided to document particularly the levels of 50 and 60 mg/m2/week, which allowed a net increase of dose intensity.

Study design Before the first course of chemotherapy, each patient had a complete medical history taken and a physical examination, complete blood cell count, measurement of renal and hepatic function parameters and serum chemistry evaluation. When a measurable tumor target was available, it was optimally defined (computed tomography and ultrasound scans, chest X-ray, magnetic resonance imaging, bone scan) before cystemustine administration. During treatment, the patients had a weekly gastrointestinal toxicity report and twice monthly biochemical profile carried out. The complete blood cell counts were repeated once a week during the first 5 weeks of the induction cycle, and twice a week during the last 3 weeks. The toxic effects were classified according to WHO criteria. Patients who received at least one cycle of treatment, i.e. doses once a week for 4 weeks followed by a 4-week rest period, were considered evaluable for toxicity. The response evaluation was also recorded according to WHO criteria. Patients were considered assessable for antitumoral activity after 8 weeks from initiation of treatment. During the subsequent cycles, the patients had a monthly response assessment.

Pharmacokinetics Sampling. Blood samples (5 ml) were obtained from a forearm vein through a teflon catheter just before drug administration and then again after 5, 15, 20, 30, 40, 50, 60, 75, 90, 120 and 180 min. Blood samples were collected in heparinized tubes previously cooled on ice, immediately transferred to cryotubes, frozen in liquid nitrogen and stored at –80°C until further analysis.

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melanomas and also against some renal carcinomas, soft tissue sarcomas and lung cancers [10–16]. This report describes the results of a new phase I trial with cystemustine administrated on a weekly schedule. The weekly regimen was chosen to evaluate the schedule-dependent pattern of toxicity, and to look for a possible improvement of clinical efficacy by partially preventing the myelosuppression, so as to increase the response rate by giving a higher total dose of cystemustine. This trial was performed at Institut Gustave Roussy (IGR) (Villejuif, France) with the aim of (i) identifying the optimal dose for further clinical trials and (ii) optimizing the results of phase II with the standard schedule. In addition, we detail the results of the blood concentration of cystemustine in patients with advanced cancer. The pharmacokinetic parameters were calculated and the relationship between the pharmacokinetics, dose level and hematological toxicity of cystemustine was evaluated. These pharmacokinetic data are important for the design of optimal therapeutic regimens to be used in subsequent clinical trials.

production of an injectable form of cystemustine was performed by Laboratoire Thissen (Braine-L’Alleud, Belgium). The drug was supplied as a dried powder in vials containing 50 mg of active drug. The vial’s contents were dissolved in distilled water <2 h before injection. The required dose was further diluted in 100 ml of 5% glucose and administered as a 15-min intravenous (i.v.) infusion of cystemustine using an Infusomat® Secura pump (B.Braun, Melsungen, Germany). The drug was given for the first chemotherapy cycle on days 1, 8, 15 and 22, followed by a 4-week rest period. When a response was obtained, patients were treated for additional cycles. Patients who had a minor response or no change continued to receive therapy at the discretion of the study chairman. Patients were treated until disease progression was documented or until unacceptable toxicity occurred.

762 Chemicals. The internal standard 3-(2-hydroxyethyl)-1,2,3-benzotriazin-4 (3H) was provided by May Becker (London, UK). Extrelut® and highperformance liquid chromatography (HPLC) solvents were obtained from Merck (Darmstadt, Germany). Drug assays. After rapid thawing, 62.5 mg of the internal standard solution in acetonitrile (2 mg/ml) was added to 2.5 ml of blood, and the extraction was carried out with dichloromethane on an Extrelut® solid phase [17].

Pharmacokinetic parameter determination. The half-life of the distribution phase (t1/2α), elimination half-life (t1/2β), area under the plasma concentration–time curve (AUC), apparent volume of distribution of the terminal phase (Vβ) and total body clearance were calculated using the extended least squares algorithm method (Siphar 4.0; Simed, Créteil, France).

Characteristic No. of patients entered

43

No. of patients evaluated for toxicity

35

Male/female

20/15

Median age, years (range)

54 (25–72)

WHO performance status 0

13

1

15

2

7

Tumor types Kidney

6

Colorectal

5

Glioblastoma-astrocytoma

5

Ovary

4

Head and neck

4

Lung

3

Mesothelium

3

Pancreas

2

Chondrosarcoma

1

Gall-bladder

1

Bladder

1

Prior treatment Surgery (%)

Pharmacodynamic evaluations. The toxic effects of the drug on platelets, granulocytes, leukocytes and hemoglobin were estimated and the AUC of the drug was related to them. The toxic (pharmacodynamic) effect was evaluated as the percentage of reduction (% Red) from initial blood cells counts: % Red = (pretreatment count – nadir count)/(pretreatment count)

No. of patients

27 (77)

Radiotherapy (%)

16 (46)

Chemotherapy (%)

28 (80)

Immunotherapy (%)

10 (29)

Chemotherapy and radiotherapy (%)

13 (37)

Prior chemotherapy

Statistical analysis Correlation between AUCs and dose or % Red were calculated by linear regression analysis; their significance was assessed using Student’s t-test on the correlation coefficient r. The Student’s t-test for paired data was applied to evaluate the difference between pharmacokinetic parameters on days 1 and 5 in the same patient. Differences of mean between two groups were analyzed by Student’s t-test, where P <0.05 was required for statistical significance.

None (%)

7 (20)

1 regimen (%)

8 (23)

2 regimens (%)

5 (14)

3 regimens (%)

5 (14)

≥4 regimens (%)

10 (29)

Total number of regimens

84

Median number of regimens (range)

2 (0–9)

WHO, World Health Organization.

Results Patient characteristics From April 1994 to December 1995, 43 patients admitted to the IGR were enrolled onto a phase I clinical trial of cystemustine. Among them, 35 patients were evaluable for toxicity. Eight (19%) were non-evaluable; two patients did no complete their cystemustine course due to complications (hospitalization in one and progression in another); and six died before completion of the first cycle.

Evaluable patient characteristics are summarized in Tables 1 and 2. Gender was almost equally represented; 57% of patients were male. The median PS was one (range 0–2), with 80% of patients presenting a PS of 0 or 1. The most frequently represented tumor types were renal (17%), brain (14%) and colorectal (14%) cancers. A variety of tumor types were present in the remaining patients. Twenty-seven patients (77%) had metastatic diseases mainly localized to the lungs

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HPLC was carried out on a Hewlett-Packard HP 1090 equipped with integrated modules. The purity of the chromatographic peaks was checked by monitoring and plotting their UV spectra (upslope, apex and downslope) measured from 200 to 400 nm. Cystemustine chromatographic analysis was conducted on a Spheri 5 silica (220 × 2.1 mm; Brownlee Labs, Santa Clara, CA) using an isocratic mode by first eluting the column for 8 min with dichloromethane–ethanol (99.5:0.5 v/v) and then with dichloromethane–ethanol (98:2 v/v) for 8 min. In this study the flow rate was 0.4 ml/min and the effluent was monitored at 230 nm. Drug concentrations were determined by calculating the peak height ratio of the drug versus internal standard and multiplying by the added concentration of internal standard (ng/ml). Calibration curves were established by plotting this parameter against the known drug concentrations, which ranged from 30 to 4000 ng/ml.

Table 1. Patient characteristics

763 Table 2. Patient characteristics by dose level Dose level (mg/m2)

Characteristics

No. of patients Assessable for toxicity

a

30

40

50

60

3

5

21

14

5

16

11

55 (43–65)

48 (41–61)

51 (23–73)

50 (29–72)

PS mean (range)

1.3 (1–2)

0.4 (0–1)

0.7 (0–2)

1.0 (0–2)

Male/female

3/0

2/3

10/11

9/5

Surgery

2 of 3

4 of 5

17 of 21

10 of 14

Radiotherapy

3 of 3

2 of 5

8 of 21

8 of 14

Chemotherapy

2 of 3

5 of 5

18 of 21

10 of 14

Immunotherapy

1 of 3

2 of 5

5 of 21

2 of 14

Chemotherapy and radiotherapy

2 of 3

2 of 5

6 of 21

7 of 14

None (%)

1 (33)

–

3 (14)

4 (29)

1 regimen (%)

1 (33)

2 (40)

1 (5)

4 (29)

2 regimens (%)

–

–

5 (24)

3 (21)

3 regimens (%)

1 (33)

1 (20)

5 (24)

1 (7)

≥4 regimens (%)

–

2 (40)

7 (33)

2 (14)

Prior treatments

Prior chemotherapy

a

Four injections completed.

Table 3. Hematological toxicity: cycle 1 (WHO criteria grade) Dose level (mg/m2)

Patients (evaluated/ entered)

Hemoglobin II III IV % III–IV

II III IV % III–IV

II III IV % III–IV

II III IV % III–IV

30

3/3

– –

–

0

1 –

–

0

1 –

1 1

40

5/5

– 2

–

40

– 1

–

20

– –

1

20

– –

1

20

50

16/21

4 1

–

6

4 5

–

31

2 6

1

44

1 5

5

63

60

11/14

7 1

1

18

2 5

4

82

4 1

6

64

– 2

6

73

White blood cells

(n = 15), nodes (n = 14) and liver (n = 10). A majority of patients had received prior chemotherapy and/or radiotherapy, although seven patients (20%) were chemotherapy naïve, including two patients who had received no prior therapy (including immunotherapy but no surgery). The 28 patients who had received prior chemotherapy had been treated with a median of two regimens (range: one to nine regimens).

Hematological toxicity Hematological toxicity was analyzed by examining 35 patients, treated with four weekly doses of cystemustine. As for most of the chloroethylnitrosourea derivatives known to date, the main toxic effect in this schedule was delayed myelosuppression. The characteristics of this hematological toxicity are clearly indicated in Table 3. The four elements [red blood

Neutrophils

–

Platelets

0

–

33

count (RBC), white blood count (WBC), hemoglobin and platelets counts] were involved. The dose-limiting toxicity was thrombocytopenia, with grade III thrombocytopenia first observed at 30 mg/m2 in a patient who had had no prior chemotherapy and a PS of 2. This toxicity was dose related both in frequency and severity (0% grade IV at 30 mg/m2, 20% at 40 mg/m2, 31% at 50 mg/m2 and 55% at 60 mg/m2). Grade IV thrombocytopenia occurred in six of 11 patients at the highest dose level (60 mg/m2), thereby defining the MTD in these groups of patients. Leucopenia and neutropenia were also common in this trial. Grade IV leucopenia and neutropenia were almost always associated with grade IV thrombocytopenia. Grade IV neutropenia was observed in 55% of patients treated at the highest dose, but only two patients were hospitalized for fever and received antibiotic therapy and

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3

Age, years [median (range)]

764

Non-hematological toxicity Non-hematological toxicities were generally mild and not dose limiting. Grade I and II nausea and/or vomiting were the most frequent non-hematological side effects. They lasted for a few hours after drug administration and were well controlled by anti-emetics, such as metoclopramide. Nausea and/or vomiting occurred in 57% of evaluable patients and were observed with a dose–effect relationship; it was observed in 0% of courses administered at 30 mg/m2, 40% at 40 mg/m2, 56% at 50 mg/m2 and 82% of courses given at 60 mg/m2. Grade III nausea and vomiting were seen in only one patient at a dose of 60 mg/m2. Infection and fever were noted in four patients (two grade I and two grade II) at the higher dose levels, 50 and 60 mg/m2, due to neutropenia (11% of cycles). One patient experienced grade III lower limb pain after infusion of cystemustine, probably related to treatment (3% of cycles). No significant liver toxicity was seen, only transient liver enzyme elevations occurred in two patients (grade I). Other

Table 4. Global responses (WHO criteria) in 31 assessable patients Response Complete Partial

No. of patients (%) 0 (0) 3 (10)

Stabilization

10 (32)

Progression

18 (58)

side effects, particularly renal, pulmonary, neurological or cardiac toxicity, alopecia or allergic reactions did not occur.

Antitumor activity Antitumor responses to cystemustine are listed in Table 4. Of the 35 patients evaluable for toxicity in this phase I study, 31 were also evaluable for response, with objective responses in three patients. A partial response was observed in one patient with lung metastasis of renal adenocarcinoma and treated at 50 mg/m2. He had previously progressed under treatment with interferon α. Two other partial responses were observed at the highest dose level (60 mg/m2). The first partial responder was a 72-year-old woman with grade IV glioblastoma. She had a volume decrease of 62% of tumor but did not receive a second treatment cycle of cystemustine due to severe hematological toxicity. The second partial responder was a 48-year-old man with brain and bone metastases of small-cell lung cancer, who had been previously treated with two lines of chemotherapy (four courses of adriamycin, etoposide, cisplatin and cyclophosphamide; two courses of etoposide and cisplatin) without response. He experienced an 81% decrease in volume of his lung lesions; however, his brain metastases had progressed after 14 weeks from the beginning of treatment, necessitating arrest of therapy. One patient with renal cancer, treated for 8 months (three treatment cycles of cystemustine), remained alive with stabilized disease.

Pharmacokinetics of cystemustine Cystemustine blood pharmacokinetics were available on day 1 of drug administration for 39 patients, with data determined on day 21 of the study for five of these patients. One was excluded from the analysis because of insufficient sampling points. Individual data, including dosing details and pharmacokinetic parameters, are listed in Table 5. Representative cystemustine blood concentrations versus times curves after a 15-min i.v. infusion have been described previously [18]. Examination of blood concentration–time profiles indicated either a monoexponential (10 data sets) or a biexponential (34 data sets) disposition. The results of the pharmacokinetic analysis showed, in the case of the biexponential model, a short half-life of the distribution phase of the intact compound (t1/2α = 3.9 min on average). The elimination half-life (t1/2β) was ∼50 min and appeared to be independent

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hematopoietic growth factors. Anemia was less frequent, grade IV occurring only in one of 36 patients. Interpatient variation in myelosuppression was also apparent at the various dose levels. The patients treated at all dose levels received multiple agents prior to cystemustine, and it was not possible to stratify them into heavily and lightly pretreated groups for comparison. However, this interpatient variability was partly related to the extent of prior treatment. Myelosuppression was delayed and reversible with a nadir in platelet counts typically occurring 5–6 weeks after initiation of the infusion (median, day 39; range 31–56), and recovery in counts usually evident 2 weeks later (median, day 49; range 41–64). The median day for the leukocyte nadir was about day 48, 9 days later than the platelet nadir. Neutropenia showed a comparable profile to leucopenia, with a median time of nadir at day 49 and recovery in the subsequent 6–11 days. For anemia, a median nadir time was also observed on day 45, and recovery time at around day 53. Increasing dose does not appear to influence the time of onset, nadir and recovery of myelosuppression. Moreover, this hematological toxicity seems to be cumulative as observed in patients who were treated with more than one drug course (four weekly doses of cystemustine followed by a 4-week rest interval) at the fixed dose. Five patients (one at 40 mg/m2, three at 50 mg/m2 and one at level 60 mg/m2) received two courses and two (one at 40 mg/m2 and one at 60 mg/m2) were treated with three courses. All exhibited a progressive decline in blood cell count, except for one patient who already had severe toxicity at the initial cycle (40 mg/m2). The cumulative effect was very clear in patients who were treated with three courses. At 50 mg/m2, one patient had no toxicity at the initial cycle, but developed grade IV thrombocytopenia, neutropenia and leucopenia associated with grade III anemia during the third cycle.

765 Table 5. Pharmacokinetic parameters (mean ± SD and variation coefficient in %) for cystemustine Dose (mg/m2)

AUC (mg.h/ml)

α

β

30

1.41 ± 0.29

2.43 ± 1.62

28.24 ± 9.33

30.96 ± 8.83

47.60 ± 13.74

n=5

20%

67%

33%

29%

29%

Half-life (min)

Vβ (l)

Total body clearance (l/h)

40

1.86 ± 0.33

6.98 ± 4.89

75.47 ± 15.45

64.77 ± 17.43

36.45 ± 10.86

n=6

18%

70%

20%

27%

30%

2.46 ± 1.06

2.93 ± 3.23

46.36 ± 23.65

40.56 ± 23.49

37.56 ± 13.97

43%

110%

51%

58%

37%

60

3.61 ± 0.90

3.84 ± 2.97

50.27 ± 28.11

38.19 ± 23.52

31.53 ± 11.42

n = 12

25%

77%

56%

62%

36%

3.87 ± 3.79

49.34 ± 25.60

42.13 ± 23.3

36.90 ± 13.41

98%

52%

55%

36%

n′ = 44

n, number of patients studied at each dose level. n′, cystemustine blood pharmacokinetics were available on the first day of drug administration for 39 patients, with data determined on day 21 of the study for five of these patients. AUC, area under blood concentration–time curve.

Figure 2. Correlation between the administered dose of cystemustine and the area under the blood drug concentration–time curve on day 1 and 22 of treatment in cancer patients. The line indicates the linear regression analysis and the Pearson correlation coefficient (r) is shown (P <0.001).

of the exponential model (P = 0.19) and of the dose (r = 0.06, P <0.68). However, t1/2β increased proportionally with increasing t1/2α (r = 0.75, P <0.0001). The apparent volume of distribution of Vβ ranged from 12 to 104 liters and remained stable as a function of dose with a mean value of 42 l (SD ± 23 l). Mean plasma clearance did not vary significantly with increased dosage and was determined at 37 ± 13 l/h. Although the interpatient variability was fairly important, cystemustine AUC increased proportionally and linearly with the dose range administered in this phase I trial (r = 0.674, P <0.0001; Figure 2).

In addition to day 1 pharmacokinetics, cystemustine kinetic profiles were also obtained on day 22 (fourth injection) in five patients. The pharmacokinetic parameters measured on the first day of infusion were not significantly different compared with day 22 (AUC: P = 0.91, Student’s paired t-test).

Pharmacokinetic–pharmacodynamic relationships Relationships between pharmacokinetic parameters of cystemustine with the main toxicities encountered in this phase I trial were assessed. For these relationships, only the first courses were used because of the possible influence of pre-

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50 n = 21

766

Discussion Cystemustine is a new CENU compound that is mostly active against human glioma and melanoma, and is at present in phase I–II evaluation. We conducted a phase I trial with cystemustine administered on a weekly schedule to establish its safety and the MTD. Indeed, this had been done quite successfully with S10036 by Godeneche et al. [18].

This study demonstrated that cystemustine is generally tolerated, with reversible toxic side effects, and can be given safely in a weekly schedule for 4 weeks followed by a 4-week rest period between courses. As expected, the hematological toxicity pattern encountered with this administration schedule was similar to that of other nitrosoureas. Myelosuppression was the major side effect and thrombocytopenia the doselimiting toxicity, although leucopenia and neutropenia were also common. These toxicities appear to be dose-related, delayed, reversible and cumulative. The MTD was 60 mg/m2, at which six of 11 patients experienced grade IV thrombocytopenia. The platelet tolerance of cystemustine was comparatively improved at 50 mg/m2, with a decrease in grade IV toxicity from 55% to 31%. On the other hand, at 40 mg/m2, only one heavily pretreated patient experienced grade IV toxicity. The tolerance of the four other patients was good, even for a patient treated with three courses (20 weeks). Thus, the recommended dose for phase II studies using this weekly schedule was 40 mg/m2. This dose could be administrated for the 16 weeks needed for drug evaluation. This dose of 40 mg/m2 for 4 consecutive weeks (cumulated dose, 160 mg/m2) represents a dose increase compared with the previous regimen using a dose of 60 mg/m2 every 2 weeks

Figure 3. Correlation between the the area under the blood drug concentration–time curve of cystemustine and the percentage decrease in (A) platelets (P <0.002), (B) hemoglobin (P <0.03), (C) leukocytes (P <0.002) and (D) granulocytes (P <0.001). The lines indicate the linear regression analysis, and the Pearson correlation coefficient (r) is shown.

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vious cystemustine administrations on the pharmacodynamics of this compound. Irrespective of the platelet level, the percentage reduction of the initial platelet count was dependent on the dose given, expressed by a linear relation between the reduction and dose (r = 0.532, P <0.002). There also was a significant relationship between the cystemustine AUC and toxic effects (r = 0.547 for platelets, P <0.002; r = 0.572 for granulocytes, P <0.001; r = 0.533 for leukocytes, P <0.002; r = 0.384 for hemoglobin, P <0.03; Figure 3). When the percentage reduction of platelets was plotted as a function of the AUCs obtained only for the patients treated at the dose of 50 mg/m2, it was found that the significant relationships observed for the whole group of patients were maintained (r = 0.525 for platelets, P <0.05).

767 Blood profiles were described by a monoexponential or a biexponential disposition, showing marked inter-individual variations in the measured pharmacokinetic parameters, as reflected by the variation coefficients (36–55%). Similar variations were also reported for this class of unstable anticancer drugs [25–27], but they were relatively low compared with other studies [28]. These variations might not be imputable to the method, which was specifically designed to avoid or minimize the compounds’ chemical decomposition during the blood collection periods [17]. The rapid elimination of intact cystemustine, with a terminal half-life of ∼50 min, was consistent with high clearance due to extensive degradation to alkylating and carbamoylating species, as with other nitrosoureas. Values ranged from very short half-lives reported for CCNU and BCNU, to longer ones found for the new CENUs, fotemustine and tauromustine [26, 29, 30]. The effect of four injections of cystemustine on the pharmacokinetic parameters were examined in five patients. The parameters measured on the first day of administration were similar to those obtained on day 22 (fourth injection), showing that cystemustine did not accumulate in blood and plasma clearance was no altered. These data suggest that the occurrence of drug metabolism acceleration or drug accumulation by repeated administration is unlikely. The Vβ was considerably larger than blood volume, indicating a good transfer of cystemustine out of blood, as with other nitrosoureas. This finding was confirmed by the ubiquitous tissue distribution of cystemustine in the rat: blood cells, liver, kidney, brain, lung, thymus, pancreas, muscle and harder gland [8]. However, the Vβ of cystemustine (42 l) was lower than that of fotemustine (61 l) [31]. This difference was probably reflected by the more intense lipophilic character of fotemustine (logP = 1.25) compared with cystemustine (logP = 0). The apparent Vβ for nitrosoureas is known to decrease with increasing hydrophilicity [32]. The dose range used in the present pharmacokinetic phase I study provides an opportunity to relate the pharmacokinetic properties of cystemustine to the dose level. The proportional increase in cystemustine AUC as a function of dose and the stability of clearance values as a function of dose demonstrated that cystemustine pharmacokinetics could therefore be considered as linear. This linearity was also reported with other nitrosoureas, particularly with TA-077 [33] and tauromustine (TCNU) [26]. Since as many as 43 patients were included in this study, we also tried to relate the pharmacokinetic properties of cystemustine to its pharmacological effects, the dose-limiting toxicity. The significant relationship between the cystemustine AUC with the percentage decrease in WBC, neutrophils and platelets may indicate that exposure to cystemustine was in part predictive for the hematological toxicity of this compound. However, other patient factors could explain interpatient variability in myelosuppression. This variability could be partly related to the characteristics of prior treatments, as patients

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(cumulated dose, 120 mg/m2). In spite of good tolerance at 40 mg/m2, the complete blood cell count should be repeated once a week for each patient. However, if a good tolerance is confirmed at 40 mg/m2 in phase II trials, a higher dose of 50 mg/m2 for the first course (4 consecutive weeks) could be explored to evaluate the possibility of better antitumor activity. Indeed, this dose (cumulated dose, 200 mg/m2) represents a dose increase compared with the previous regimen using a dose of 90 mg/m2 every 2 weeks (cumulated dose, 180 mg/m2). Moreover, the preliminary results of this study showed antitumor responses in the highest dose range administered (50–60 mg/m2). Nevertheless, in this trial the observed thrombocytopenia (63% of grade III–IV) was of increased importance relative to the previous phase II trial (42% of grade III–IV thrombocytopenia in melanoma [14]). The difference in toxicity between the two regimens can be partly attributed to differences in patient characteristics (patients heavily pretreated in phase I). Therefore, the optimal treatment could be a first course at a dose of 50 mg/m2, followed by a second course at a dose of 40 mg/m2 to limit the cumulative toxicity of cystemustine. Non-hematological toxicity was mainly limited to nausea and vomiting, which were generally mild and dose dependent. From prior experience with nitrosourea analogs, potential problems existed with pulmonary [19, 20], chronic renal [21, 22] and hepatic [23, 24] toxicity. Careful monitoring of liver enzymes and creatinine revealed no evidence of acute damage of these organs. No specific tests for pulmonary toxicity were monitored other than clinical status and chest X-rays. However, no patient exhibited evidence of hypoxia or shortness of breath. No other side effects were observed, particularly neurological or cardiac toxicity, alopecia or allergic reactions. These observations agree with other communications or published data on cystemustine [9–16]. After repeated functional tests for fibrosis, four long-term survivors (>5 years) treated with this drug have not shown evidence of pulmonary, hepatic or renal toxicity. However, one has presented a mild dysmyelopoiesis and one a secondary leukemia 10 years after treatment. The results of the present pharmacokinetic study are in accordance with a previous phase II pilot trial that consisted of injection of cystemustine 60 or 90 mg/m2 every 2 weeks [18]. Similar results were observed for pharmacokinetic parameters: between half-lives reported for this trial and the previous phase II trial (mean values of 50 and 45 min, respectively); between volumes of distribution (mean value of 42 ± 23 and 38 ± 13 l, respectively) and between total clearance (mean value of 37 ± 13 and 35 ± 8 l/h, respectively). Concerning cystemustine AUC, our data indicate that there was no difference between the two regimens at a dose of 60 mg/m2 (3.6 ± 0.9 mg.h/ml for the weekly schedule and 3.0 ± 0.6 mg.h/ml for the previous regimen). These pharmacokinetic parameter comparisons between two different trials confirmed the good reproducibility of this method, which uses fast freezing, solid phase extraction and HPLC analysis.

768

Acknowledgements The authors thank Fabrice Kwiatkowski for his help with the statistical analysis and Bénédicte Annoble, Sophie Amat, Christine Rolhion and Nathalie Martineau for helpful discussion. This work was supported in part by Ligue Nationale Contre le Cancer (Comité du Puy de Dôme) and by a Cystemustine grant (Program Hospitalier de Recherche Clinique, Paris, France).

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treated at all dose levels received multiple agents prior to cystemustine, and it was not possible to stratify them into heavily and lightly pretreated groups for comparison. Alkylation at the O6 position of guanine has been thought to be important in the cytotoxicity of nitrosoureas [34, 35]. The ability of cells to repair this lesion using the enzyme O6-alkylguanineDNA alkyltransferase (MGMT) appears to be a principal determinant of cytotoxicity to these agents [36, 37]. MGMT levels in hematopoietic cells were not assayed in this study. However, interpatient variability in myelosuppression may be due to differences in the baseline MGMT levels or progenitor damage related to prior chemotherapy. Whether or not there is a correlation between these pharmacokinetic parameters with the antitumor activity of cystemustine remains to be established in phase II trials, but it is noteworthy that in this phase I trial, the antitumor responses were observed in the highest dose range administered (50–60 mg/m2), where the highest AUC was observed. Also, if activity (especially in glioma) and a dose–response relationship were confirmed in future trials, then the possible use of hematopoietic growth factors could facilitate the administration of high doses in patients. In addition, in the future, depletion of MGMT activity could theoretically potentiate the cytotoxicity of cystemustine. In previous work, we have shown that administration of O6-benzyl-N2-acetylguanosine (BNAG), which is a direct substrate for MGMT, enhances the therapeutic index of cystemustine on nude mice bearing resistant human melanoma [38]. Another approach has recently given a greater emphasis to the development of new nitrosoureas with sufficient clinical tolerance, i.e. the perspectives of potentiation of antitumor activity by methionine deprivation [39, 40]. This could constitute a new approach for potentiation, if clinically achievable. In summary, it appears from this study that the analytical method has good reproducibility. Moreover, the significant relationship between cystemustine AUC and the percentage decrease of WBC, neutrophils and platelets may indicate that exposure to cystemustine was in part predictive for the hematological toxicity of this compound. A reasonable starting dose for phase II studies is 40 mg/m2, with dose escalation based on blood cell counts.

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