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Evaluation of In Vitro Drug Interactions with Karenitecin, a Novel, Highly Lipophilic Camptothecin Derivative in Phase II Clinical Development

From the Division of Pharmacy (J. A. Smith, T. Madden), Division of Cancer Medicine, Department of Gynecologic Medical Oncology (J. A. Smith), and the Pharmaceutical Development Center (R. A. Newman, T. Madden), University of Texas M.D. Anderson Cancer Center, Houston, Texas, and BioNumerik Pharmaceuticals, Inc., San Antonio, Texas (F. H. Hausheer).

Address for reprints: Timothy Madden, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 90, Houston, TX 77030.

	ABSTRACT

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The objective of this study was to describe the potential metabolism and protein-binding interactions with karenitecin, a novel computer-engineered, highly lipophilic camptothecin. Individual cloned cytochrome P450 (CYP450) isoenzymes were used to determine, in vitro, the metabolism of karenitecin. Known substrates and inhibitors of each isoenzyme were employed to evaluate CYP450 drug interactions with karenitecin. To assess the extent, variability, and role of various drug-binding proteins, the authors examined, in vitro, the effects of both albumin (Alb) and -acidic glycoprotein (AAG) on karenitecin plasma protein binding (PPB). Equilibrium dialysis techniques were used to measure the free fraction of karenitecin in the presence of varying ratios of Alb and AAG. Artificial plasma, spiked with karenitecin, was dialyzed for 72 hours at 37°C against a Sorensen's buffer solution using regenerated cellulose membranes having a molecular weight cutoff of 12 to 14 kDa. Additional protein-binding experiments were conducted to assess the potential PPB drug interactions between karenitecin and other highly protein-bound drugs commonly used in the treatment of cancer patients. In vitro experiments suggested that karenitecin is metabolized by CYP450 3A4, 2C8, and 2D6 isoenzymes and is an inhibitor of the CYP450 3A4 and 2C8 isoenzymes. The mean (± SD) percentage of karenitecin bound to plasma proteins was 99.1% ± 0.27%. The extent of karenitecin protein binding was directly proportional to the plasma concentration of AAG. Protein-binding displacement interactions were observed in the in vitro experiments with phenobarbital, phenytoin, mitoxantrone, and salicylic acid. It was concluded that karenitecin has the potential to alter CYP450 3A4 and 2C8 drug-metabolizing activity. In addition, in vitro PPB evaluations have demonstrated that karenitecin may displace other highly PPB drugs and that slight variations in plasma AAG concentration may result in large variations in free drug exposure. Each of these interactions could potentially result in increasing the toxicity or alter the efficacy of combination anticancer drug therapy if they are significant in patients. Future karenitecin clinical trials should include studies to monitor or evaluate the effects of these potential drug interactions on the overall toxicity of karenitecin when used in combination with other drugs.

Key Words: Karenitecin • camptothecin • plasma • urine • pharmacokinetics • pharmacodynamics • metabolism • protein binding • anticancer agent • oncology

Clinical trials in the early 1970s of the plant product camptothecin, formulated in sodium hydroxide to enhance solubility, were terminated due to a lack of clinical antitumor activity and a significant incidence of hemorrhagic diarrhea and uroepithelial toxicity.⁴ These unexpected toxicities are believed to have been due to the administration of camptothecin (which exists in solution as a pH-dependent mixture of lactone and carboxylate forms) as its water-soluble but biologically inactive carboxylate salt.

Medicinal chemists interpreted these observations as an opportunity and initiated efforts to discover improved camptothecin analogs. The most popular approach was preparation of analogs with aqueous solubility, adequate for convenient IV administration, at pH values predominantly favoring the active lactone form of the molecule. These efforts have been successful, culminating recently in regulatory approvals of two semisynthetic compounds, topotecan and irinotecan (CPT-11).^5-8 While the success of the water-solubilizing approach to camptothecin analog development is apparent, lipophilic compounds might also possess new and useful pharmacologic or pharmacokinetic properties relative to water-soluble compounds/prodrugs. Modulation of these properties, for example, might enhance a compound's therapeutic efficacy or safety or change its anticancer spectrum.

Karenitecin, 7-[(2-trimethylsilyl)ethyl]-20(S)camptothecin, is the first highly lipophilic camptothecin derivative. It was designed using molecular modeling to engineer a compound with more desirable characteristics: superior potency, absence of complex metabolic activation, and enhanced stability of the active lactone form.⁹ Extensive modeling, examining multiple side chain moieties in a variety of positions, led to the final proposed compound, karenitecin, having a silane moiety at position 7 on the B ring (Figure 1). Preclinical pharmacology and toxicology studies confirmed the relative safety and efficacy of karenitecin, leading to its rapid introduction into clinical trials. We conducted the following in vitro metabolism experiments to investigate the following properties of karenitecin: (1) the presence or absence of metabolism by certain cytochrome P450 isoenzymes, (2) whether this metabolism was affected by known inhibitors of these isoenzymes, and (3) whether karenitecin could act as an inhibitor of any of these isoenzymes. The in vitro data presented here describe the initial investigations into the metabolism and plasma protein binding of this agent in humans.

View larger version (9K): [in this window] [in a new window]   Figure 1. Structure of karenitecin (7-[(2-trimethylsilyl)ethyl]-20(S)camptothecin).

	METHODS

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Cytochrome P450 Metabolism Experiments
In vitro evaluation of karenitecin metabolism by the CYP450 isoenzymes 3A4, 2D6, 2C8, and 2C9 was completed using isolated human cytochrome P450 proteins for each isoform (Gentest Corporation, Woburn, MA). The assay cofactor, buffer, and dilution solutions were prepared fresh for each experiment. Briefly, the cofactor solution was prepared with nicotinamide adenine dinucleotide phosphate (20 mg/mL), glucose-6-phosphate (20 mg/mL), magnesium chloride hexahydrate (133 mg/mL), and deionized water (to a final volume of 10 mL). The assay dilution/buffer solution used for all isoenzymes, except for the CYP2D6 experiments, was prepared with 1.5 mL potassium phosphate (0.5 M solution), 1.5 mL cofactor solution (as above), 0.3 mL glucose-6-phosphate dehydrogenase (G6PDH, 40 U/mL), and 11.7 mL deionized water. The 2D6 assay dilution/buffer solution was prepared similarly, except only 0.188 mL of the cofactor solution and 13 mL of deionized water were used.

Appropriate substrates and inhibitors were used for each isoenzyme, and samples were incubated for 45 minutes at 37°C. Reactions were halted with enzyme-specific stopping solutions (Table I) and then placed on ice until analysis. Samples were analyzed by high-performance liquid chromatography (HPLC) methods described previously.^10-15 For each experiment, control samples with a known amount of substrate and synthesized metabolite, in the absence of the isoenzyme, were prepared for qualitative comparisons. All experiments were performed in triplicate.

Protein-Binding Experiments
Protein-binding equilibrium dialysis methods were used. Briefly, Sorensen phosphate buffer was prepared from analytical-grade reagents. Fresh, drug-free human plasma was obtained from normal volunteers just prior to the start of each experiment. Regenerated cellulose membranes with a 12- to 14-kD molecular weight cutoff (MWCO) were used in each dialysis macro-well (Spectrum Laboratories, Rancho Dominguez, CA). Five plasma samples were spiked at each of the following karenitecin concentrations: 100, 250, 500, and 1000 ng/mL. After completing the appropriate time analysis studies, samples were rotated in a dialysis incubator for 48 hours at 37°C to reach equilibrium. Experiments were repeated in triplicate.

To assess the variability and role of drug binding, we examined, in vitro, the effects of both albumin (Alb) and -acidic glycoprotein (AAG) concentration on karenitecin plasma protein binding (PPB). Again, equilibrium dialysis techniques were used to measure the free fraction of karenitecin in the presence of varying ratios of Alb and AAG. Buffer solutions containing varying concentrations of both Alb and AAG were spiked with 50 ng/mL karenitecin and dialyzed for 72 hours at 37°C against a Sorensen's solution using cellulose membranes with an MWCO of 12 to 14 kD (Table II). All experiments were performed in quadruplicate.

To evaluate whether protein-binding displacement drug interactions might occur with coadministration of karenitecin and other highly protein-bound drugs, we examined the effect of nine compounds commonly used in patients with cancer on karenitecin PPB. The agents evaluated were phenobarbital, phenytoin, dexamethasone, salicylic acid, quinidine, lidocaine, gemcitabine, and mitoxantrone. Equilibrium dialysis techniques were used to determine the free fraction of karenitecin in the presence of each of these other agents. Fresh plasma from a consenting normal volunteer was spiked with each agent at clinically relevant concentrations and 50 ng/mL karenitecin and then dialyzed for 72 hours at 37°C.

	RESULTS

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Cytochrome P450 Metabolism
In vitro experiments to assess whether karenitecin is a substrate for any of the four CYP450 isoforms most associated with xenobiotic metabolism—3A4, 2C8, 2C9, and 2D6—were performed. These experiments demonstrated that karenitecin is metabolized by the 3A4, 2C8, and 2D6 isoenzymes, with approximately 50% being metabolized in the short 45-minute incubation period. This was comparable to the extent of metabolism observed in the known substrates of each isoenzyme for this time interval. In addition, the metabolism of karenitecin was inhibited by both ketoconazole, a known inhibitor of CYP450 3A4, and sulfaphenazole, a known inhibitor of both CYP450 2C8 and 2C9. Moreover, karenitecin itself demonstrated inhibition of CYP450 isoenzymes 3A4 and 2C8, thus showing the drugs' potential to inhibit its own metabolism (Figure 2a,b).

View larger version (69K): [in this window] [in a new window]   Figure 2. (a) Cytochrome P450 3A4 (50 µg protein): substrate = testosterone (165 µg/mL), inhibitor = ketoconazole (0.25 mM), karenitecin 150 ng/mL. Samples were incubated for 45 minutes at 37°C. Data are presented as mean ± SD of triplicate determinations. Cytochrome P450 2C8 (500 µg protein): substrate = paclitaxel (213 µg/mL), inhibitor = sulfaphenazole (0.5 mM), karenitecin 100 ng/mL. Samples were incubated for 45 minutes at 37°C. Data are presented as mean ± SD of triplicate determinations. (b) Cytochrome P450 2D6 (100 µg protein): substrate = bufuralol (25 µM), inhibitor = quinidine (25 µM), karenitecin 200 ng/mL. Samples were incubated for 45 minutes at 37°C. Data are presented as mean ± SD of triplicate determinations. Cytochrome P450 2C9 (500 µg protein): substrate = diclofenac (100 µM), inhibitor = sulfaphenazole (0.5 mM), karenitecin 200 ng/mL. Samples were incubated for 45 minutes at 37°C. Data are presented as mean ± SD of triplicate determinations.

Protein-Binding Experiments
Protein-binding studies confirmed karenitecin to be highly protein bound, having a mean percentage protein bound of 99.1% ± 0.27%. Protein binding did not appear to be concentration dependent, with the mean percentage bound ranging from 98.8% to 99.3% at the five karenitecin concentrations (100-1000 ng/mL) evaluated.

After demonstrating that karenitecin had binding affinity for both Alb and AAG alone, the effect of altering the ratio of Alb to AAG concentration was explored (Table II). The overall binding of karenitecin increased as the AAG to Alb ratio increased. These data suggest that while Alb and AAG both have a role in karenitecin PPB, variability in AAG concentration may result in greater changes in the karenitecin lactone free fraction since it appears that karenitecin has a greater binding affinity for AAG.

Another factor that could result in an extensive change in the karenitecin lactone free fraction is binding displacement interactions with other highly protein-bound drugs. Of the nine drugs evaluated, combinations of karenitecin with phenytoin, mitoxantrone, and salicylic acid resulted in twofold increases in karenitecin free drug concentration. The largest of these displacement effects occurred with phenobarbital, resulting in a fourfold increase in the free fraction of karenitecin (Figure 3). The other camptothecin analogs were not evaluated in these plasma protein-binding experiments, so it is not known at this time whether these drugs display similar interactions.

View larger version (31K): [in this window] [in a new window]   Figure 3. Karenitecin plasma protein-binding (PPB) displacement. Bar graphs demonstrate the mean percentage karenitecin lactone protein bound when coincubated with the respective drug. Data are presented as means of triplicate determinations. The following drug concentrations were used: dexamethasone, 25 µg/mL; phenytoin, 15 µg/mL; salicylic acid, 25 µg/mL; phenobarbital, 20 µg/mL; quinidine, 3 µg/mL; lidocaine, 2 µg/mL; gemcitabine, 30 µg/mL; and mitoxantrone, 100 ng/mL.

	DISCUSSION

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The development of camptothecin derivatives has been challenging. Among the problems encountered in the development process has been the significant and unpredictable nature in the pharmacokinetics and pharmacodynamics of these compounds. While there are numerous reasons for this observed variability, the three most important are the relative water insolubility of these agents, significant differences in their metabolism, and the relative instability of the active lactone form of these compounds.

Camptothecins undergo a reversible, rapid, pH-dependent hydrolysis of the active closed-ring lactone form to the inactive open-ring carboxylate form. The inactive carboxylate form binds to human serum albumin, preventing conversion back to the active lactone form, thus limiting pharmacologic activity. Burke et al¹⁶ and Bon et al¹⁷ observed that modifications at the 7 or 9 position of the quinolone nucleus increase the binding affinity of the carboxylate form to albumin, thereby lowering the plasma lactone concentration. However, our clinical observations and in vitro experiments suggest that albumin binding does not reduce or exert any clinically important effect on the lactone stability of karenitecin. In addition, AAG appears to be more important in the plasma protein binding of karenitecin, and as observed with Alb, binding to AAG does not appear to reduce the lactone stability of karenitecin.

We have analyzed the clinical pharmacologic and pharmacodynamic data from the initial phase I study of karenitecin in humans (data not shown). Data from that study established that, as observed in the clinical trials of other camptothecin derivatives, karenitecin pharmacokinetic behavior shows substantial interpatient variability when comparisons are made between dose levels. However, the interpatient pharmacokinetic variability within the cohort treated at the phase II recommended dose (the largest in that study) was substantially smaller. While these observations could be due to the nearly uniform dose administered to these patients and the large number of patients at this dose level, other factors, including smaller interpatient differences in metabolism and protein binding, could contribute to this reduction in pharmacokinetic variability.

We investigated the plasma protein-binding characteristics of karenitecin and found that this drug is similar to other classes of newly introduced oncology drugs. The degree of karenitecin plasma protein binding appears to be influenced more by AAG than Alb.¹⁸ In addition, we found that karenitecin was subject to plasma protein-binding displacement when combined with other highly protein-bound drugs, thereby greatly increasing the free fraction of karenitecin. With a mean PPB in human subjects of 99%, these data demonstrate that slight variations in plasma AAG concentration may result in large variations in free drug exposure. On the basis of the current information from two phase I trials, we do not see evidence of significant displacement in patients, but we intend to monitor for this and other potentially important drug interactions closely in the future. What may be more important than drug displacement is drug sequestration that might result in a reduction in the amount of drug reaching the target. One recent report suggests that AAG may sequester large amounts of drug, thereby rendering an otherwise active drug inactive. Gambacorti-Passerini et al,¹⁹ using a mouse model, demonstrated in vivo that high levels of AAG, associated with the level of tumor burden, reduced the amount of unbound STI-571 available to the target, resulting in reduced drug efficacy. Since karenitecin plasma protein binding is as high as 99% in clinical samples, increases in AAG during therapy may reduce the effectiveness of this agent.

In vitro drug metabolism studies demonstrated that karenitecin was subject to metabolism by CYP450 3A4, 2C8, and 2D6 isoenyzmes. In addition to being a substrate for these isoenzymes, karenitecin appears to be an inhibitor of CYP450 3A4 and 2C8. Karenitecin, therefore, may have the potential to inhibit its own metabolism. Caution should be exercised in subsequent clinical trials to carefully monitor patients who are receiving other medications that are substrates of CYP450 3A4 or 2C8. The chemical composition and possible clinical activity of karenitecin metabolites have yet to be fully delineated and is currently the subject of further investigation in our laboratory.

Karenitecin does have the potential for both plasma protein binding and CYP450-mediated drug interactions that could be clinically important. Further studies are needed to determine the structure of the metabolites and possible activity of any karenitecin metabolites. Those studies and clinical evaluations of an oral formulation of karenitecin and dose-intense IV regimens, as well as studies examining the utility of karenitecin with other anticancer agents, are ongoing.

Karenitecin is a novel, highly lipophilic camptothecin derivative that, unlike other camptothecins, predominates in the plasma in the active lactone form at physiological pH. This is a significant advantage since the stability of the active lactone form of other camptothecin derivatives is reported to be < 50%. In addition, the plasma half-life of karenitecin is up to 10 times greater than that reported for other camptothecin derivatives.²⁰ These characteristics suggest that the pharmacokinetic profile of karenitecin, compared to other camptothecin analogs, may be less variable, thereby making it a more predictable camptothecin for clinical use. These pharmacokinetic characteristics may have an additional benefit by yielding sustained cytotoxic activity since karenitecin persists longer and appears to remain in its active lactone form for an extended interval. It is important to consider that many of these observations for karenitecin may also be observed for other camptothecins; this is an area of interest to us for future development.

	FOOTNOTES

 
DOI: 10.1177/0091270003255921

Submitted for publication October 10, 2002; Revised version accepted May 7, 2003.

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