9 Clinical ExperienceWith 9-Aminocamptothecin

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Camptothecins in Cancer Therapy

Edited by: V. R. Adams and T. G. Burke © Humana Press Inc., Totowa, NJ 193

Clinical Experience

With 9-Aminocamptothecin

Lessons for New Drug Development

Chris H. Takimoto, MD , P h D

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1. INTRODUCTION

In preclinical studies first published in 1989 by Giovanella and col- leagues, 9-aminocamptothecin (9-AC) demonstrated highly promising anti- tumor activity against human colorectal cancer xenografts (1). This led to an extensive drug development program for 9-AC initially under the guidance of the National Cancer Institute, and later in cooperation with pharmaceu- tical industry sponsors. However, despite this impressive preclinical activ- ity, 9-AC has not demonstrated clinically useful antitumor activity to date.

Thus, in contrast to its more successful brethren, irinotecan and topotecan,

9-AC is no longer under active clinical development. Nonetheless, testing

of other novel and new camptothecin derivatives remains an active area of

pharmacological research. Thus a careful review of the early clinical and

pharmacological trials of 9-AC may help us to understand why 9-AC has not

shown more meaningful antitumor activity and may also provide important

lessons for the future drug development of novel camptothecins and other

topoisomerase I (TOP-I) targeting agents.

2. HISTORICAL BACKGROUND

In 1986, Drs. Wani and Wall first synthesized the water-insoluble camptothecin derivative, 9-AC (2). Subsequent laboratory studies revealed that 9-AC was a potent inhibitor of TOP-I (3,4), and its ability to interfere with this enzyme correlated with its in vivo cytotoxic activity against murine L1210 leukemia cells (3,5). In 1989, Giovanella and colleagues published their impressive results in the journal Science documenting the activity of 9-AC in human colon cancer xenografts models in nude mice (1).

9-AC was highly active with minimal systemic toxicity and generated better antitumor responses than a number of different anticancer agents including 5-fluorouracil, doxorubicin, melphalan, methotrexate, vincristine, vinblas- tine, and others (1). Interest in clinically developing this compound was further heightened by the demonstration of a broad range of activity against a diverse group of human tumor xenografts including malignant melanoma

prostate (10), breast (11), and bladder cancers (12).

In animal studies, the principal dose-limiting toxicities of 9-AC were neutropenia and gastrointestinal toxicity. Preclinical pharmacological stud- ies also revealed that brief intravenous infusions generating high peak con- centrations for short periods were toxic and relatively inactive against human tumor xenografts. Instead, subcutaneous administration of 9-AC as a sus- pension in Tween-80 resulted in sustained plasma concentrations of 9-AC lactone in the nanomolar range and generated antitumor responses (13).

3. CLINICAL DEVELOPMENT

In 1989, the US National Cancer Institute selected 9-AC as a high-prior- ity compound for further clinical development. However, clinical trials with this agent were delayed because of difficulties in formulating this com- pound resulting from poor aqueous solubility. In 1993, 9-AC was success- fully formulated in dimethylacetamide, polyethylene glycol, and phosphoric acid for use in phase I clinical trials. Later, in 1995, Pharmacia & Upjohn developed a newer colloidal dispersion formulation of 9-AC that was supe- rior in stability and in compatibility with standard intravenous infusions.

This newer colloidal dispersion formulation has been used in the more recent clinical studies of this agent.

3.1. Phase I Trials

Because preclinical studies highlighted the importance of prolonged

exposures to 9-AC lactone, the first phase I trials of 9-AC dimethylacetamide

used a continuous 72-hour intravenous infusion administered every 2 (14)

or every 3 (15,16) weeks (Table 1). Both schedules yielded similar toxicity

Table 1 9-Aminocamptothecin (9-AC) Phase I Clinical Trials 9-AC Recommended phase II regimenformulationPrincipal toxicitiesReference 0.84 mg/m2/day CIV × 3 days every 2 weeks, orDMANeutropenia14 1.13 mg/m2/day CIV × 3 days every 2 weeks with G-CSFNeutropenia and some thrombocytopenia 1.08 mg/m2/day CIV × 3 days every 3 weeksDMANeutropenia and some thrombocytopenia15 1.3 mg/m2/day CIV × 3 day every 3 weeksCDNeutropenia16 0.48 mg/m2/day CIV × 5 days weekly for 3 of 4 weeksDMANeutropenia17 Diarrhea 0.6 mg/m2/day CIV × 5 days weekly for 2 of 3 weeksCDNeutropenia 0.4 mg/m2/day CIV × 7 days every 4 weeksDMANeutropenia and some thrombocytopenia51 0.51-0.6 mg/m2/day CIV × 7 days every 4 weeksCD 1.65 mg/m2 CIV over 24 hours weekly for 4 of 5 weeksCDNeutropenia18 1.1 mg/m2/day IV over 30 minutes daily × 5 days every 3 weeksCDNeutropenia19 Oral Preparations No recommended oral dose daily × 5 every 2 weeksCD (oral)No dose-limiting toxicity determined20 0.84 mg/m2 orally daily × 14 days every 3 weeksPEG-1000Myelosuppression and diarrhea21 Specialized phase I studies Pediatric patients 1.25 mg/m2/day CIV × 3 days every 3 weeksDMANeutropenia Thrombocytopenia26 Acute leukemia1.4 mg/m2/day CIV over 7 daysCDMucositis27 IV, intravenous; CIV, continuous intravenous infusion; G-CSF, granulocyte-colony-stimulating factor; DMA, dimethylacetamide; CD, colloidal dispersion.

profiles with dose escalation limited by neutropenia and, less commonly, thrombocytopenia. Other toxicities included mild to moderate fatigue, ane- mia, nausea and vomiting, diarrhea, alopecia, and mucositis. In contrast to irinotecan, severe diarrhea was not observed on the 72-hour infusion sched- ules. Additional schedules of 9-AC administration tested in a phase I clini- cal trials include prolonged 120-hour infusions given weekly for 2 or 3 consecutive weeks followed by a week rest period (17) and a weekly 24- hour infusion given for 4 of every 5 weeks (18) (Table 1). For most of these regimens, the major dose-limiting toxicity was still myelosuppression;

however, diarrhea became more pronounced when prolonged administra- tion schedules were tested (17).

More recently, a short 15-minute infusion given daily for 5 days every 3 weeks was developed for clinical testing (19). On this schedule, neutropenia and thrombocytopenia were dose limiting and the recommended phase II dose was 1.1 mg/m

/day infused over 15 minutes daily for 5 days every 3 weeks. The colloidal dispersion formulation of 9-AC has also been admin- istered to patients orally over a dose range of 0.2 to 0.68 mg/m

/day daily

mined because of large interpatient variability in the area-under-the-con- centration versus time curve. Another oral study used 9-AC formulated as a polyethylene glycol 1000 capsule with much greater success (21). Admin- istration of 9-AC capsules over a dose range of 0.25 to 1.5 mg/m

/day for 14 days every 3 weeks resulted in a recommended phase II dose of 1.1 mg/

m

/day. Neutropenia and thrombocytopenia were dose limiting, and the oral bioavailability of 9-AC in capsular form was a relatively consistent 48

17.8%.

3.2. Clinical Pharmacology

All camptothecin derivatives, including 9-AC, contain a terminal lactone

ring that rapidly hydrolyzes in aqueous solutions to form the more hydro-

philic open-ring carboxylic acid species. Under nonacidic conditions, the

equilibrium for this reaction very much favors a less active carboxylate

form. The pharmacokinetics of the lactone and carboxylate species of 9-AC

have been extensively examined (Table 2). Overall, the amount of active

lactone 9-AC circulating in plasma is quite low relative to the total (lactone

+ carboxylate) 9-AC, with most studies reporting a lactone-to-total 9-AC

plasma ratio of less than 0.16. This value is lower than the active lactone

forms of topotecan (0.16–0.2) (22) or irinotecan (0.51) (23,24) that circulate

in plasma. In addition, the plasma half-life for 9-AC lactone species is

relatively short, ranging from 4.5 to 10 hours (18,19,25–27). Urinary excre-

tion of unchanged drug ranges from 8.6 to 32.1% of the administered dose

Table 2 9-Aminocamptothecin Clinical Pharmacology Volume ofPercent Terminaldistribution atUrinarylactone in half-lifesteady-stateCLexcretionplasma Dose and scheduleFormulationCompound(hours)(L/m2)(L/hour/m2)(%)(%)Reference 0.12–1.8 mg/m2/dayDMALactone4.47 ± 0.53195 ± 11424.5 ± 7.3NR8.7 ± 4.725 IV over 72 hoursTotal8.38 ± 2.123.6 ± 10.6NR32.1 ± 8.3 (over 24 hours) 1.08 mg/m2/day IVDMALactone42.2 ± 34.412.6 ± 3.655.2 ± 18.0NRNR15 over 72 hours 0.86–1.49 mg/m2/dayDMALactone7.1 ± 3.5135.3 ± 52.5NRNR10.8 ± 3.626 IV over 72 hours*Total8.1 ± 3.821.2 ± 13.3NRNR 0.7–1.9 mg/m2/dayCDLactone10.7 ± 6.7111.0 ± 72.318.0 ± 9.0NR7.9918 IV over 24 hours 0.9–1.55 mg/m2/dayCDLactone22.5 ± 8.5325 ± 14530.3 ± 4.5NRNR16 IV over 72 hours 0.45–1.4 mg/m2/dayCDLactone4.3 ± 2.758.4 ± 23.311.3 ± 4.9NRNR27 CIV over 7 days 0.48 mg/m2/day CIVDMALactone NRNR12.3 ± 7.1NR13.817 over 5 daysTotal NRNR1.65 ± 0.87NR 0.7–1.9 mg/m2/dayCDLactone10.7 ± 6.7111.0 ± 72.318.0 ± 9.0NR7.9918 IV over 24 hoursTotal7.7± 4.617.7 ± 6.02.36 ± 1.248.6 ± 4.0NR IV, intravenous,; CIV, continuous intravenous infusion; DMA, dimethylacetamide formulation; CD, colloidal dispersion formulation; NR, not reported. *Pediatric patients.

In a pharmacological study of 9-AC in brain cancer patients, subjects receiving anticonvulsant medications tolerated much higher doses of 9-AC consistent with an increased clearance of drug (28). One possible explana- tion is that 9-AC hepatic clearance may be increased by enzyme induction caused by antiepileptic drugs such as phenytoin, carbamazepine, phenobar- bital, primidone, felbamate, and valproic acid. However, hepatic metabo- lites of 9-AC have not been characterized (29). An alternative explanation is that these same enzyme inducing antiepileptic agents may also result in the increase expression of transport proteins leading to increased 9-AC clearance and biliary excretion (30).

Large interpatient variation in the plasma kinetics of 9-AC has been consistently observed in most studies, which is similar to clinical pharma- cological studies of other camptothecin derivatives such as topotecan (31) and irinotecan (23,24). In pharmacodynamic studies, moderate to strong correlations between dose-limiting myelosuppression and 9-AC steady state plasma concentrations (25,30) or overall area under the concentration-time curve (AUC) (18,19,32) have been observed.

3.3. Antitumor Activity

The antitumor activity of 9-AC administered as a 72-hour infusion has been examined in a variety of different tumors (Table 3). In phase II studies in advanced colorectal cancer, administration of 9-AC at 1.2 to 1.4 mg/m

/ day over 72 hours every 2 weeks to 16 previously untreated patients resulted in no objective responses (33). However, the myelosuppressive toxicity of this regimen was substantial, with grade 4 neutropenia seen in 56% of patients and febrile neutropenia documented in 31% (33). No objective antitumor activity was also seen in another trial examining 9-AC administered at 0.84 mg/m

/day over 72 hours every 2 weeks in 17 untreated colorectal cancer patients (34). In contrast, in advanced platinum refractory ovarian cancer patients, 72-hour infusions of 9-AC were active generating response rates of 21% (35) and 19% (36). Activity in relapsed or refractory lymphoma patients was also observed (37). Administration of 9-AC at 0.96 mg/m

over 72 hours every 3 weeks with granulocyte colony-stimulating factor to 40 patients with advanced lymphoma resulted in a partial response rate of 25%

(95% confidence interval [CI] 13–41%) and a median survival of 12.5 months. In advanced cutaneous T-cell lymphoma, 9-AC at 1.1 mg/m

/day every 2 weeks with granulocyte colony-stimulating factor resulted in partial responses in 2 of 12 patients for an overall rate of 17%, but myelosuppressive toxicity was high and 3 patients died from neutropenia and sepsis (38).

Minimal activity has also been reported in non-small-cell lung, central nervous system, and breast cancers (Table 3).

More prolonged infusion schedules administering 9-AC over 120 hours weekly for 3 of 4 weeks have also been tested in a limited number of studies.

In 17 previously untreated advanced colorectal cancer patients, administra-

tion of 0.48 mg/m

/day for 120 hours weekly for 3 of 4 weeks again resulted in no objective responses (39). In lung cancer, administration of 120-hour infusions of 9-AC to stage IIIB and IV non-small-cell lung cancer patients generated only a single response in 12 previously untreated patients and produced a 1-year survival rate of 28% (95% CI = 5–58%) and a median survival of 10.2 months (40).

The rights to this agent were transferred in 1997 from the Pharmacia &

Upjohn Company to the IDEC Pharmaceutical Corporation. In this same year, phase II testing of 9-AC was initiated using a 1.1 mg/m

intravenous infusion daily for a 5-day schedule (19). The results of phase II testing of 9- AC on this schedule have not been formally reported; however, in 1999 IDEC notified the US government that it was stopping the development of 9-AC based on preliminary phase II results (41). Thus, despite showing impressive activity in preclinical models of colorectal cancer, 9-AC did not produce clinically meaningful antitumor activity of 9-AC in patients with this disease. Modest activity has been observed in refractory lymphoma patients and in women with platinum-refractory ovarian cancer, but the overall phase II activity of 9-AC on any schedule has been largely disap- pointing.

4. LESSONS FOR NEW DRUG DEVELOPMENT This lack of clinically useful antitumor activity raises several key ques- tions. First, why were the preclinical animal xenograft studies of 9-AC so promising initially given the later clinical findings? Second, what pharma- cological characteristics make camptothecin derivatives such as topotecan and irinotecan so much more clinically successful than 9-AC?

The first question was explored in detail in an interesting study by Kirstein et al. (42). Careful analysis of the pharmacological characteristics of 9-AC in nude mouse experiments were compared with the clinical pharmacologi- cal findings seen in early human studies of 9-AC. They adopted a novel approach first suggested by Minderman and colleagues (43). This involved rigorously defining the minimally effective dose of 9-AC that caused objec- tive tumor regressions in animals (MEDOR). However, Kirstein and col- leagues expanded the MEDOR concept to include a minimally effective threshold exposure to 9-AC in plasma defined in terms of the AUC, thereby generating a new parameter that could more easily be applied across species to preclinical and clinical studies. The administration of 9-AC daily for 5 days for 2 consecutive weeks generated a MEDOR AUC of 690–1580 ng/

mL/hour in the nude mouse experiments (42). The mouse MEDOR AUC

was then compared with the AUC profiles generated in humans in Phase I

clinical trials. Unfortunately, dose-limiting myelosuppressive toxicity in

humans limited the 9-AC AUC to a substantially lower range of 126–493

ng/mL/hour. The greater sensitivity of human bone marrow to 9-AC limited

Table 3 9-Aminocamptothecin Phase II Clinical Trials PreviousMedianMedian therapy forduration ofduration of EvaluatedadvancedResponse rateResponseresponsesurvival Tumor type9-AC regimenpatientsdisease (%) type(months) (months)Reference Colorectal1.42 mg/m2/day IV for 72 hours14Yes0 (95% CI 0–20)0 PRNR9.533 every 2 weeks + GCSF(95% CI 7.3–12.9) Colorectal0.84 mg/m2/day IV for 72 hours17No00 PRNRNR34 every 2 weeks Colorectal0.48 mg/m2/day IV for 12017No00 PRNR8 (0.75–14.5)39 hours weekly for 3 of 4 weeks Nonsmall-cell lung1.42 mg/m2/day IV for 72 hours54No9 (95% CI 3–19)5 PR2.25–7.05.452 every 2 weeks + GCSF Nonsmall-cell lung0.6 mg/m2/day IV for 120 hours12No8 (95% CI 0–21)1 PRNR10.240 weekly for 2 of 3 weeks Refractory Hodgkin’s0.96 mg/m2/day IV for 72 hours40Yes25 (95% CI 13–41)10 PR5 (r 1–10)12.537 and non-Hodgkin’severy 3 weeks ± G-CSF lymphoma Central nervous0.85–1.78 mg/m2/day IV39NR31 PRNRNR53 systemfor 72 hours + 2 weeks Head and neck0.85–1.0 mg/m2/day IV14No00 PRNR6 (1–9+)54 for 72 hours + 2 weeks

Ovarian0.84–1.13 mg/m2/day IV28Yes216 PRNRNR35 for 72 hours + 2 weeks with GCSF Ovarian0.84 mg/m2/day IV for 72 hours27Yes19 (95% CI 6–38)5 PRNRNR36 + 2 weeks Breast1.1 mg/m2/day IV for 72 hours15Yes132 PR3.5-5NR55 every 2 weeks + GCSF Cutaneous T-cell1.1 mg/m2/day IV for 72 hours12Yes172 PRNR538 lymphomaevery 2 weeks +GCSF IV, intravenous; G-CSF, granulocyte-co;ony stimulating factor; PR, partial response; NR, not reported; CI, confidence interval.

the plasma AUC in humans to levels below the minimally effective expo- sures range required for optimal anticancer activity. Even modest decreases in achievable systemic exposure can mean the difference between success and failure for drugs, such as the camptothecins, that have steep dose response curves and narrow therapeutic indices. This analysis is also quite consistent with previous findings showing that mouse bone marrow pro- genitor cells are 6 to 11 times more resistant to 9-AC than their human counterparts (44). These studies predict, a priori, that maximal achievable concentrations of 9-AC in plasma would be lower in humans compared with mice because of marked differences in host tolerance to toxic drug effects.

Kirstein and colleagues recommended that careful pharmacokinetic and pharmacodynamic studies be performed both in preclinical experiments and in early clinical trials. Better communication between preclinical scien- tists and physicians designing and conducting phase I clinical trials may further help to streamline the drug development process.

The second question of which pharmacological characteristics of topotecan and irinotecan make them more clinically useful than 9-AC is difficult to answer with certainty. Differences in the clinical pharmacology of these camptothecin derivatives are quite prominent. For example, both irinotecan and topotecan are much more water-soluble than 9-AC and are easier to formulate in pharmaceutical preparations. Furthermore, the lac- tone forms of both of these camptothecins are more stable than 9-AC in plasma with higher percentage of the active lactone species circulating in plasma after intravenous administration (25). For topotecan it is 16–20%

(22), irinotecan (SN-38) 51–64% (23,24), and for 9-AC approximately 9%

(25). The tight protein binding of the open ring 9-AC carboxylate to plasma

albumin, which tends to destabilize the lactone species (45), may explain this difference. In contrast, the protein binding of topotecan is much less, and for SN-38, plasma protein binding interactions actually tend to favor the stability of the active lactone species (46). Another kinetic difference is found in the half-lives of these agents. The half-life of SN-38 is relatively long, 11 ± 3 hours, whereas for topotecan it is 3.01 ± 0.54 hours and for 9- AC it is 4.5 ± 0.5 hours. For this reason, irinotecan can be given as a rela- tively short infusion over 90 minutes every 3 weeks (23,24), whereas topotecan and 9-AC are administered on consecutive days or as prolonged infusions over many days.

Additional differences at the molecular level of action may also be impor-

tant in distinguishing between the different camptothecin analogues. For

example, SN-38 is highly potent as an inhibitor of TOP-I and generates

cleavable complexes in colon cancer cells that are more stabile than those

generated by 9-AC or topotecan (47). Molecular resistance mechanisms

may also differ. P-glycoprotein-associated multidrug resistance may have

some impact on the activity of some of these agents. Multidrug resistant

(MDR)-expressing cell lines are ninefold more resistant to topotecan and twofold more resistant to 9-AC (48) compared with parental wild-type cells.

In contrast, irinotecan and SN-38 are not effective substrates for the MDR drug efflux pump (49) and cross-resistance between irinotecan and vincris- tine or doxorubicin is not observed in P388 leukemia cells expressing the MDR phenotype (50).

In summary, 9-AC is more similar to topotecan than irinotecan. For example, both 9-AC and topotecan are not a prodrugs, they are a weak MDR substrates and both have relatively short half-lives in plasma after intrave- nous administration. Topotecan and 9-AC both exhibit dose-limiting toxici- ties consisting of myelosuppression rather than diarrhea and both have shown activity in ovarian cancer. Consequently, 9-AC was simply not unique enough to justify its further clinical development as a novel TOP-I- targeting agent.

5. CONCLUSIONS

The great promise of 9-AC seen in preclinical studies has not resulted in clinically useful antitumor activity. The reasons for this failure have been reviewed and provide some insights into further drug development in this area. However, there are no easy answers. Fundamentally, we still need to have a much better understanding of the pharmacology of these agents.

Important and as-yet poorly understood issues include how these different camptothecins function at the molecular level, what are their key determi- nants of antitumor response, and what are their clinically relevant mecha- nisms of drug resistance? Despite these challenges, there is no doubt that TOP-I is a validated molecular target for anticancer therapy and the future drug development efforts are sure to result in new generations of useful and better agents that target this enzyme.

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