High antimetastatic efficacy of MEN4901/T-0128, a novel camptothecin carboxymethyldextran conjugate.

The antimetastatic activity of a novel camptothecan conjugate, MEN4901/T-0128, in which 7-ethyl-10-aminopropyloxy-camptothecin (T-2513) is bound to a biodegradable carboxymethyldextran via a Gly-Gly-Gly linker, was observed in this study. High antimetastatic activity of MEN4901/T-0128 was demonstrated in a clinically-relevant orthotopic mouse model of human colon cancer. MEN4901/T-0128 and irinotecan were compared for anti-metastatic activity as well as efficacy against the primary tumor. An imageable, metastatic model was made by surgical orthotopic implantation (SOI) of the green fluorescent protein (GFP)-expressing HT-29 tumor in nude mice. MEN4901/T-0128 and irinotecan were administered intravenously at various doses and schedules. MEN4901/T-0128, with treatment beginning on d 49 after SOI, was highly effective on lymph node metastasis as well as against the primary tumor. Both GFP imaging and histology demonstrated a markedly lower metastatic incidence of lymph nodes in all MEN4901/T-0128 treated mice compared with irinotecan-treated and untreated mice. At the most efficacious dose of MEN4901/T-0128, only 1 of 12 animals had lymph node metastasis compared with 19 of 20 in the control group. The present study demonstrates the principle that when a camptothecan is conjugated to an appropriate polymer, the drug can become extremely effective with important clinical potential for antimetastatic therapy, a most urgent need.

Antitumor activity of delimotecan against human metastatic melanoma: pharmacokinetics and molecular determinants.

Delimotecan (MEN 4901/T-0128) is a new cytotoxic prodrug constituted by a camptothecin analog (T-2513) bound to carboxymethyl dextran through a triglycine linker. A significant antitumor activity of delimotecan against human metastatic melanoma xenograft model Me15392 is reported. Dacarbazine, the drug approved for the treatment of metastatic melanoma, was ineffective in this melanoma model. Pharmacokinetic studies, together with the expression analysis of mRNA for enzymes involved in delimotecan metabolism, showed that T-2513 and other cytotoxic metabolites of delimotecan (SN 38 and T-0055) are generated in greater quantities in the tumor tissue than in toxicity target tissues, such as liver, thus accounting for the antitumoral activity. Moreover, we demonstrated that human metastatic melanoma cells are able to phagocytose delimotecan and cleave it to release the cytotoxic moieties T-2513 in the tumoral environment. Further flow cytometric analysis showed a higher recruitment of macrophages in xenografted human metastatic melanoma, when compared with other human tumors. Thus, the antitumoral activity of delimotecan exerted on metastatic melanoma is due to several factors: (i) the ability of melanoma cells to phagocytose and metabolise delimotecan; (ii) the accumulation of delimotecan in tumoral mass; (iii) the recruitment of macrophage cells to the melanoma nodule and (iv) the expression in melanoma cells of a pattern of enzymes that converts delimotecan into cytotoxic metabolites. Based on these results, delimotecan might be exploited as a new anticancer agent for the therapy of metastatic melanoma because of its high efficacy and good selectivity, and therefore clinical trials for this indication are warranted.

Clinical and pharmacologic study of the novel prodrug delimotecan (MEN 4901/T-0128) in patients with solid tumors.

PURPOSE: To investigate i.v. administration of delimotecan (MEN 4901/T-0128), a carboxymethyldextran polymer prodrug of the active camptothecin derivative T-2513, and to assess the maximum tolerated dose, safety profile, clinical pharmacology, and antitumor activity of delimotecan and metabolites. EXPERIMENTAL DESIGN: Patients with solid tumors refractory to standard therapy received i.v. delimotecan as 3-hour infusion once every 6 weeks. The starting dose was 150 mg/m2, followed by an accelerated dose escalation with at least one patient per dose level. The pharmacokinetics of delimotecan, T-2513, and its metabolites, SN-38, SN-38G, T-1335, T-0055, and T-3921, were assessed in plasma and urine, and their pharmacodynamics were determined by measuring the effect of the treatment on hematologic and nonhematologic toxicity. RESULTS: Twenty-two patients received 35 courses. Dose-limiting toxicities were observed at 5,400 mg/m2 (n = 1), 3,600 mg/m2 (n = 1), and 2,400 mg/m2 (n = 2). The dose level of 1,800 mg/m2 was determined as maximum tolerated dose. Two partial responses were observed in patients with anal cancer (1800 mg/m2) and head and neck cancer (2400 mg/m2). Delimotecan had a long terminal half-life of 109 h, and relatively high exposures to T-2513 and SN-38 were obtained. The percentage decrease in WBC and absolute neutrophil count significantly correlated with the dose of delimotecan. CONCLUSIONS: Based on its preliminary antitumor activity, safety profile, and pharmacokinetic profile, we recommend to evaluate delimotecan given as 3-hour infusion once every 6 weeks at a dose level of 1,800 mg/m2 in a phase II study.

Single-molecule observations of topotecan-mediated TopIB activity at a unique DNA sequence.

The rate of DNA supercoil removal by human topoisomerase IB (TopIB) is slowed down by the presence of the camptothecin class of antitumor drugs. By preventing religation, these drugs also prolong the lifetime of the covalent TopIB-DNA complex. Here, we use magnetic tweezers to measure the rate of supercoil removal by drug-bound TopIB at a single DNA sequence in real time. This is accomplished by covalently linking camptothecins to a triple helix-forming oligonucleotide that binds at one location on the DNA molecule monitored. Surprisingly, we find that the DNA dynamics with the TopIB-drug interaction restricted to a single DNA sequence are indistinguishable from the dynamics observed when the TopIB-drug interaction takes place at multiple sites. Specifically, the DNA sequence does not affect the instantaneous supercoil removal rate or the degree to which camptothecins increase the lifetime of the covalent complex. Our data suggest that sequence-dependent dynamics need not to be taken into account in efforts to develop novel camptothecins.

Integrated computational strategies for UV/vis spectra of large molecules in solution.

In recent years, the margin of interaction between computational chemistry and most branches of experimental chemistry has increased at a fast pace. The experimental characterization of new systems relies on computational methods for the rationalization of structural, energetic, electronic and dynamical features. In particular, novel computational approaches allow accurate estimates of molecular parameters from spectroscopic optical observables, giving rise to synergic interactions between experimentalists and theoretically-oriented chemists. Our main objective in this tutorial review is to delineate the degree of advancement of possible integrated computational approaches to the interpretation of optical spectroscopies, with an accent on large molecules in solvated environments, based on the combination of advanced quantum mechanical treatments and stochastic modelling of relaxation processes.

Human and murine macrophages mediate activation of MEN 4901/T-0128: a new promising camptothecin analogue-polysaccharide conjugate.

MEN 4901/T-0128 is a new cytotoxic prodrug constituted by the camptothecin analogue T-2513 bound to carboxymethyl dextran through a triglycine linker. MEN 4901/T-0128 was designed to target the active camptothecin at the tumour site. MEN 4901/T-0128 is weakly cytotoxic in vitro and thus T-2513 must be released from the conjugate to become active. Here, we demonstrated that human purified cathepsin B releases T-2513 from MEN 4901/T-0128 at pH values ranging from 3 to 5. pH dependency of this reaction suggests that cleavage of the linker should mainly occur in the lysosomes. As elevated cathepsin B activity has been described in macrophages, human tumour monocytic THP-1 cells differentiated into macrophage-like cells were used to study the cellular mechanisms responsible for MEN 4901/T-0128 antitumour activity. Here, we show that differentiated THP-1 internalizes MEN 4901/T-0128 efficiently in a time-dependent and concentration-dependent manner. After phagocytosis, THP-1 cells can cleave the prodrug and release T-2513 in the media. On the contrary, undifferentiated THP-1 cells or pancreatic ASPC-1 tumour cells, although expressing high levels of cathepsin B, are much less efficient in the release of cytotoxic moieties in the culture media. Moreover, normal murine macrophages, recovered from the peritoneal cavity or from the spleen, when activated (in vitro by 100 ng/ml phorbol 12-myristate-13-acetate and in vivo by 300 microl of 3% w/v thioglycollate solution), were able to release (after incubation with 10 microg/ml MEN 4901/T-0128) cytotoxic moieties in the culture supernatant, in an amount sufficient to kill human carcinoma A2780 cells. Thus, we suggest that tumour-associated macrophages may play a key role in the uptake of MEN 4901/T-0128, cleavage and local release of active moiety T-2513. This mechanism should support a tumour targeting of the cytotoxic moieties, allowing an improved antitumour efficacy/safety ratio for MEN 4901/T-0128.

MEN4901/T-0128, a new camptothecin derivative-carboxymethyldextran conjugate, has potent antitumor activities in a panel of human tumor xenografts in nude mice.

PURPOSE: The purpose of the present study was to evaluate the antitumor activity and pharmacokinetic profile of MEN4901/T-0128 in nude mice bearing human tumor xenografts in comparison with irinotecan (CPT-11) and T-2513. EXPERIMENTAL DESIGN: We have determined the antitumor activity of MEN4901/T-0128, CPT-11, and T-2513 in BALB/cA Jcl nude mice bearing human gastric (H-81), colon (H-110), lung (Mqnu-1, H-74), esophageal (H-204), liver (H-181), and pancreatic (H-48) cancer lines, which had been serially transplanted s.c. and maintained in nude mice, and characterized the pharmacokinetic profile of MEN4901/T-0128 in nude mice bearing human gastric carcinoma St-4. RESULTS: MEN4901/T-0128 administered i.v. showed a marked antitumor activity in each of these tumor models, producing tumor shrinkage in the models of H-204 and H-181 carcinomas at its maximum tolerated dose of 80 mg/kg (expressed as T-2513) weekly for 4 weeks (q7d x 4) and tumor-shrinking or marked growth-inhibitory effects in the models of H-81, H-110, Mqnu-1, H-74, and H-48 carcinomas at 1/3 of its maximum tolerated dose (q7d x 4). Pharmacokinetic analysis showed that MEN4901/T-0128 had an extended plasma half-life with sustained tumor levels of T-2513, which may explain the superior activity of MEN4901/T-0128 in vivo. CONCLUSIONS: Because the efficacies of some drugs in this human cancer-nude mouse panel correlated well with their clinical outcomes in patients with the same type of cancers, the findings provide direct support that MEN4901/T-0128 is more efficacious than CPT-11 and is an excellent candidate for clinical trials for the treatment of solid tumors.

Macromolecular and nanotechnological modification of camptothecin and its analogs to improve the efficacy.

Camptothecin (CPT) and its analogs are some of the most potent antitumor agents known. However, their poor water-solubility and high toxicity require changes of their physicochemical and biological characteristics. Active lactone forms have provoked interest in the utility of CPT and its analogs again. Macromolecular chemical modifications and nanotechnological formulations have been used to obtain improved systems of CPT-related compounds. In these systems, one of the most important concepts is the enhanced permeability and retention (EPR) effect. The outcomes obtained by these approaches are displayed by introducing concrete examples.

[Synthesis and antitumor activity of 20-O-linked camptothecin ester derivatives].

AIM: To improve the profile of 20 (S)-camptothecin, a series of 20-O-linked camptothecin phenoxyacetic acid ester derivatives have been designed. METHODS: These derivatives were synthesized by the method of acylation. Their chemical structures were confirmed with 1HNMR, IR, MS, and HRMS. The cytotoxicities of the compounds were tested by MTT assay. The in vivo antitumor activities of these esters were evaluated against mouse liver tumor H22 in mice. RESULTS: Twelve derivatives of camptothecin ester are new compounds. CONCLUSION: In vitro and in vivo antitumor activity has indicated that some derivatives appeared significantly more effective than topotecan in the H22 mouse liver tumoral model.

Determination of plasma topotecan and its metabolite N-desmethyl topotecan as both lactone and total form by reversed-phase liquid chromatography with fluorescence detection.

Topotecan (TPT) undergoes hepatic N-demethylation forming N-desmethyl topotecan (NDS). To evaluate the effect of drug-drug interactions on NDS disposition in children receiving TPT we developed and validated a sensitive and specific HPLC-fluorescence detection method for lactone and total (lactone plus carboxylate) TPT and NDS. Deproteinized plasma is vortexed, centrifuged, and the methanolic extract diluted with water for the lactone form of NDS and TPT or diluted with 1.5% phosphoric acid for NDS and TPT total. A 100 microL sample is injected onto a Varian ChromGuard RP column attached to an Agilent SB-C(18) reversed-phase analytical column held at 50 degrees C. The mobile phase (flow-rate, 0.8 mL/min) consists of methanol-aqueous buffer (27:73, v/v) (75 mM potassium phosphate and 0.2% triethylamine, pH 6.5). TPT and NDS were detected with excitation and emission wavelengths set at 376 and 530 nm, respectively. The standard curves for both forms of TPT ranged from 0.25 to 80 ng/mL, and for NDS ranged from 0.10 to 8.0 ng/mL. Within-day and between-day precision (% RSD) was

Carrier and dose effects on the pharmacokinetics of T-0128, a camptothecin analogue-carboxymethyl dextran conjugate, in non-tumor- and tumor-bearing rats.

T-0128 is a novel camptothecin (CPT) analogue (T-2513: 7-ethyl-10-aminopropyloxy-CPT)-carboxymethyl (CM) dextran conjugate via a Gly-Gly-Gly linker, with a molecular weight (MW) of 130 kDa. Our previous studies demonstrated that T-0128 has strong antitumor activity against human tumor xenografts that are highly refractory to CPT analogues attributable to the passive tumor targeting of released T-2513. This study examines the effects of carrier, dose, and tumor on T-0128 pharmacokinetics. To study carrier effect, tumor-bearing rats received one i.v. injection of fluorescein isothiocyanate (FITC)-labeled CM dextran with a different degree of substitution (DS) of the carboxymethylated groups and a different MW. Results showed that CM dextran from Dextran T-110 (MW 110 kDa) with a DS value of 0.4 is an appropriate drug carrier for T-0128 regarding plasma half-life and passive tumor targeting. To study dose and tumor effects, non-tumor- and tumor-bearing rats were treated with T-0128 doses ranging from 1 to 25 mg/kg (based on the amount of T-2513 bound to CM dextran). Dose-dependent pharmacokinetics of T-0128 were observed in both kinds of rats. The presence of tumor reduced the plasma half-life and systemic exposure of T-0128. The saturation of hepatic and splenic tissue uptake clearances (CLups), and a large contribution of the tumor CLup to the total body clearance explain these results. Overall, our data provide a rationale for the selection of the carrier for T-0128 and a need for pharmacokinetic studies to evaluate the influences of tumor on the drug disposition.

Macrophage-mediated activation of camptothecin analogue T-2513-carboxymethyl dextran conjugate (T-0128): possible cellular mechanism for antitumor activity.

Camptothecin (CPT) analogue T-2513-carboxymethyl (CM) dextran conjugate (T-0128) suppressed human tumor xenografts that were refractory to CPTs. This improvement was explained by its altered pharmacokinetics, but the cellular mechanism of action is still not clear. For this reason, in the present study we examined the determinants of T-0128 action at the cellular level. In vitro tests showed that T-0128 was inactive, indicating that the requirement for its activity lies in the release of linked T-2513, accompanied by the cellular uptake of the conjugate. The accumulation varied between cell lines: tumor cells, including Walker-256 carcinoma and B16 melanoma, showed only a marginal uptake and an undetectable drug release in the medium. In contrast, macrophage-like cells, such as J774.1, internalized T-0128 very efficiently, and liberated T-2513. With regard to the mode of accumulation, fluid-phase pinocytosis seems to be a key factor based on the followings: a similar cell-specificity existed in the uptake of FITC dextran, a marker of fluid-phase pinocytosis. Also, the macrophage uptake of T-0128 increased almost linearly with its medium concentration and was insensitive to dextran sulfate, a ligand for macrophage scavenger receptor. Comparative efficacy studies of T-0128 in the presence and absence of macrophages demonstrated that macrophages increased the efficacy of T-0128. The enhancement could be explained in terms of increases in the amount of released T-2513. Overall, these results lead us to the conclusion that T-0128 acts like a Trojan horse with the help of macrophages: T-0128 is taken up by macrophages in tumor tissues, and the liberated T-2513 kills tumor cells.

Determinants for the drug release from T-0128, camptothecin analogue-carboxymethyl dextran conjugate.

To improve pharmacological profiles of camptothecins (CPTs), a new macromolecular prodrug, denoted T-0128, was synthesized. This prodrug comprises a novel CPT analog (T-2513: 7-ethyl-10-aminopropyloxy-CPT) bound to carboxymethyl (CM) dextran through a Gly-Gly-Gly linker, with a molecular weight of 130 kDa. The present study was designed to elucidate the mechanisms that promote the release of linked T-2513. First, we compared the abilities of a rat liver homogenate, a cocktail of its lysosomal enzymes, and different types of pure enzymes, to liberate T-2513 from the conjugate. The releasing rate in the homogenate was very slow, but was accelerated with the lysosomes. Lysosomal cysteine proteinases, such as cathepsin B, were responsible, coupled with the results of in vitro and in vivo inhibition studies using proteinase inhibitors. The pH optimum for the cathepsin B-mediated drug release was approximately 4. This corresponds to the pH in lysosomes, suggesting lysosomotropic release. Second, to assess the effect of the length and composition of the peptidyl linker, we synthesized the conjugates with a different linker and compared the drug-releasing rates. We found that the insertion of Phe into Gly-Gly-Gly allowed various kinds of enzymes to produce a rapid cleavage, and the Gly-chain lengthening enhanced the lysosome-mediated drug release. The released T-2513 levels in the liver and tumor of the tumor-bearing rats dosed with each conjugate increased with the length of Gly linker, suggesting a good in vitro to in vivo relationship. Comparative efficacy studies of the conjugates with a different linker demonstrated that T-0128 showed the maximum efficacy against MX-1 human mammary xenograft tumors. Thus the Gly-Gly-Gly linker exploits lysosomal cathepsin B to liberate T-2513 slowly and steadily, resulting in improved therapeutic efficacy.

Complete regression of xenografted human carcinomas by camptothecin analogue-carboxymethyl dextran conjugate (T-0128).

Clinically available camptothecins (CPTs), such as irinotecan (CPT-11) and topotecan, represent one of the most promising classes of antitumor agents, despite their toxicity. To improve their pharmacological profiles, a new macromolecular prodrug, denoted T-0128, was synthesized. This prodrug is a novel CPT analogue (T-2513)-carboxymethyl (CM) dextran conjugate via a triglycine spacer, with a molecular weight of Mr 130,000. This study was designed to test the concept that the rational design of a CPT-polymer conjugate would increase the efficacy of the parent drug. The in vivo antitumor study against Walker-256 carcinoma demonstrated that T-0128 was 10 times as active as T-2513, supporting this concept. Additionally, comparative efficacy studies of T-0128, T-2513, CPT-11, and topotecan were performed using a panel of human tumor xenografts in nude mice, showing the advantage of T-0128 over these CPTs. The maximal tolerated doses (MTDs) of T-0128, T-2513, and CPT-11 were comparable. Even a single i.v. injection of T-0128 at 6 mg/kg (based on the amount of T-2513 bound to CM dextran) induced complete regression of MX-1 mammary carcinoma. T-0128 at 10 mg/kg weekly for 3 weeks (one-tenth of its MTD) cured LX-1 lung carcinoma. Also, T-0128 below its MTD consistently cured or regressed St-4 gastric and HT-29 colorectal tumor xenografts that are highly refractory to CPTs. These demonstrate the broad range of therapeutic doses achieved with T-0128. Pharmacokinetic studies were performed to correlate the efficacy results obtained for T-0128 with plasma and tissue drug concentrations using Walker-256 tumor-bearing rats. Results showed that after i.v. administration of T-0128, the conjugate continued to circulate at a high concentration for an extended period, resulting in tumor accumulation. In the tumor, the sustained release of T-2513 occurred. In contrast, T-2513 disappeared rapidly from the body. The significant increases in the amount and exposure time of released T-2513 in the tumor explain well the enhanced efficacy of T-0128. In conclusion, this study indicated that T-0128 improved the potency of T-2513, demonstrating the proof of the above concept.

Quantification of topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces by high-performance liquid chromatographic methods.

Sensitive high-performance liquid chromatographic (HPLC) methods have been developed and validated for the simultaneous determination of the antitumor drug topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces. Both compounds are reversibly hydrolysed to their hydroxycarboxylate forms at physiologic pH. Separate HPLC systems have been developed for the determination of lactone and total (lactone plus hydroxycarboxylate forms) concentrations in plasma. The instability of the analytes in plasma requires immediate protein precipitation with ice-cold methanol. The lactone forms of the analytes were stable in the methanol extracts for at least 15 months when stored at -70 degrees C. For the determination of the total levels, the plasma extracts were acidified with 25 mM phosphoric acid to convert the compounds into their lactone forms quantitatively. The sample pretreatment procedure for urine included dilution in methanol while the faecal samples were homogenized in distilled water and then extracted twice with an acetonitrile-ammonium acetate mixture. Separation was achieved on reversed-phase columns (Zorbax SB-C18) and detection was performed fluorimetrically at 380/527 nm. Within-run and between-run precisions were less than 10% and average accuracies were between 90 and 110%. The methods were used in a mass balance study in patients with malignant solid tumors to determine the disposition and routes of elimination of topotecan and N-desmethyltopotecan.

O-glucuronidation, a newly identified metabolic pathway for topotecan and N-desmethyl topotecan.

During topotecan analysis of clinical urine samples, an additional peak eluting just after the solvent front was observed. This potential metabolite was isolated by chromatographic methods. Mass spectrometry data along with chromatographic retention data and fluorescence characteristics showed that the isolated fractions contained two compounds, i.e. topotecan-O-glucuronide and N-desmethyl topotecan-O-glucuronide. The concentrations of the metabolites in human urine were relatively low. When topotecan was given as a 30 min infusion at a dosage of 1.5 mg/m2 daily for five consecutive days every 3 weeks, the maximal metabolite concentrations in a 24 h urine sample were approximately 10% of topotecan-O-glucuronide and 3.5% of N-desmethyl topotecan-O-glucuronide with respect to the concentration of topotecan in the urine. This is the first report demonstrating that glucuronide metabolites of topotecan are present in the urine of treated patients.

Formation of a novel topotecan metabolite in the hormone-independent human prostate carcinoma cell lines DU-145 and PC-3.

To investigate the implications of drug metabolism on topotecan (TPT) resistance in prostate cancer cells, we measured the time-dependent uptake, metabolismrand efflux of TPT in the prostate cancer-derived cell lines DU-145 and PC-3 by HPLC. Exposure of DU-145 to 10 microM TPT resulted in a maximal intracellular concentration of TPT of 12.6 +/- 0.53 pmol/10(6) cells (t = 10 min) with a decrease to 4.4 +/- 0.25 pmol/10(6) cells after 2 hours. Incubation of PC-3 cells, however, revealed a more than 2-fold higher level of cytoplasmatic TPT (25.3 +/- 4.8 pmol/10(6) cells). In both cell lines, an intracellular metabolite was detectable after 30 minutes. Its concentration continuously increased reaching saturation after 6 hours (0.015 +/- 0.003 pmol/10(6) cells in DU-145 and 0.0059 +/- 0.0020 pmol/10(6) cells in PC-3 cells). Analysis of the culture supernatant of DU-145 and PC-3 cells revealed that this metabolite is secreted into the medium at increasing concentrations (0.220 +/- 0.025 and 0.079 +/- 0.008 pmol/10(6) cells, respectively). In accordance with the elevated formation of the TPT-metabolite in DU-145 cells, the expression of cytochrome P450 (CYP) isoenzymes CYP3A, CYP2B, CYP2D and CYP2E as measured by Western blot analysis was also higher in this cancer cell line. In conclusion, we found that TPT is rapidly taken up by the two prostate cancer cell lines and metabolized to a minor biotransformation product dependent on their content of cytochrome P450 isoenzymes. The structural identification of this TPT metabolite and the CYP isoenzyme(s) responsible for its formation remain to be elucidated.

Phenytoin alters the disposition of topotecan and N-desmethyl topotecan in a patient with medulloblastoma.

Topotecan undergoes both renal and hepatic elimination, with topotecan urinary recovery ranging from 60 to 70%. We evaluated the potential of phenytoin to alter the disposition of topotecan and its N-desmethyl metabolite. A 5-year-old child with high-risk medulloblastoma received the first course of topotecan with phenytoin and the second course without phenytoin. For both courses, topotecan doses were adjusted to achieve a target topotecan lactone plasma area under the curve (AUC). Serial plasma samples were obtained, and lactone and total plasma concentrations of topotecan, as well as total plasma and cerebrospinal fluid concentrations of N-desmethyl topotecan, were measured by high-performance liquid chromatography. Phenytoin coadministration increased lactone and total topotecan clearance from 43.4 +/- 1.9 L/h/m2 to 62.9 +/- 6.4 L/h/m2, and 20.8 +/- 2.8 L/h/m2 to 30.6 +/- 4.1 L/h/m2, respectively (P < 0.05). Concomitant phenytoin increased the plasma AUC of total N-desmethyl topotecan from 7.5 +/- 0.68 ng/ml x h to 16.3 +/- 0.53 ng/ml x h (P < 0.05) at plasma AUC of total topotecan of 226.0 +/- 5.5 ng/ml x h and 240.9 +/- 39.8 ng/ml x h, respectively. N-Desmethyl topotecan penetrated into the cerebrospinal fluid (0.12 +/- 0.01). The patient experienced no grade 3 or 4 toxicity. These are the first data documenting altered topotecan and N-desmethyl topotecan disposition when coadministered with phenytoin and suggests that topotecan may undergo further hepatic metabolism. Although there is an increase in exposure to the active N-desmethyl topotecan metabolite, it is less than the decrease in exposure to topotecan lactone. Therefore, patients concomitantly administered phenytoin may require an increase in topotecan dose to achieve a similar pharmacological effect as a patient not receiving phenytoin.